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The arrival of Mary in England created a great difficulty for Elizabeth. If she were allowed to escape to France, both France and Spain might join hands to enforce her claims to the English succession, and if she were restored to the throne of Scotland, Moray and his friends could expect no mercy. It was determined, therefore, that Elizabeth should act as umpire between the queen and her rebellious subjects, so that by inducing both sides to submit their grievances to Elizabeth feeling between them might be embittered, and that in the meantime a divided Scotland might be kept in bondage. In her reply to the letter received from the Queen of Scotland Elizabeth informed her that she could not be received at court nor could any help be given to her unless she had cleared herself of the charges brought against her. Both parties in Scotland were commanded to cease hostilities, but at the same time Cecil took care to inform Moray secretly that he should take steps to enforce his authority throughout Scotland.[30]

Mary, while repudiating Elizabeth's right to sit in judgment on her conduct, consented that a conference should be held between her commissioners and those appointed by Elizabeth and by the rebel lords.

The Dukes of Norfolk, Sussex, and Sir Ralph Sadler were the English commissioners; Bishop Leslie, Lord Livingstone, and Lord Herries represented Mary; while Moray, Morton, and Maitland of Lethington appeared to present the case of the rebel lords. The conference opened at York (October 1568). Several days were wasted in attempts made by Maitland to effect a compromise so that the production of charges and counter-charges might be unnecessary, and in considering inquiries put forward by the Earl of Moray regarding Elizabeth's attitude in case the charges against the Scottish queen were proved. Some of the letters supposed to have been written by Mary to Bothwell were shown secretly to the English commissioners, but they do not seem to have produced any great effect on the Duke of Norfolk or even on the Duke of Sussex who was certainly not prejudiced in Mary's favour. The latter reported that Moray could produce no proofs except certain letters the authorship of which the Queen of Scots would deny. In fact, Sussex believed that were the affair to come to trial it would go hard with the queen's accusers.[31] In a short time Elizabeth ordered that the venue should be changed from York to London, and Mary, believing that she would be allowed an opportunity to defend herself before the peers and representatives of foreign governments, accepted the change. She sent Bishop Leslie and Lord Herries to represent her in London, but on their arrival they found that Mary would not be allowed to appear in person, though her accusers were received by the queen, nor would the foreign ambassadors be admitted to hear the evidence.

The new commission opened at Westminster (4th Dec. 1568). The lords brought forward their charges against the queen accusing her of complicity in the murder of her husband. In proof of this they produced a number of letters that were supposed to have been contained in a casket left behind him by Bothwell in Edinburgh, when he fled from that city in June 1567. This casket contained eight letters and some sonnets, which, if really written by Mary, proved beyond doubt that she was hand in glove with Bothwell in bringing about the murder of Darnley. The Casket Letters considered in the light of her own conduct furnished damaging evidence of Mary's guilt. Whether these letters were genuine or forged is never likely to be established with certainty,[32] but considering the character of Mary's opponents, their well-known genius for duplicity, the contradictory statements put forward by their witnesses and the indecent haste with which the whole enquiry was brought to a close, it is difficult to believe that the evidence of Mary's authorship was convincing. The commissioners acting on Mary's behalf laboured under grave disadvantages from the fact that their mistress was not at hand for consultation. As a consequence they made many mistakes in their pleadings, but they were on sure ground when they demanded that copies of the incriminating letters should be forwarded to Mary for examination. This demand, though supported by the French ambassador, was refused, and Mary was never allowed an opportunity to reply to the main charge brought against her. An offer was made that proceedings should be dropped if Mary would consent to resign the throne of Scotland in favour of her son, and when she refused this offer the conference was brought to a sudden termination. Moray and his friends were informed that "nothing had been produced against them as yet that might impair their honour and allegiance; and on the other part there had been nothing sufficiently produced or shown by them against the queen their sovereign, whereby the Queen of England should conceive or take any evil opinion of the queen her good sister for anything yet seen" (Jan.

1569).[33] The Earl of Moray and his companions were allowed to return to Scotland, and nothing more was done either to establish the innocence or the guilt of the Queen of Scotland. The object of Elizabeth and her advisers had been attained. They had blackened the character of Mary; they had driven a wedge between herself and her nobles, and had allowed Moray to return to Scotland to rule as an English dependent.