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 Lettres de Catherine de Médicis, vol. ii.). In the spring of 1564 he was engaged in negotiating at Troyes a peace with France, and found, as he conceived, his chief obstruction in the conduct of his colleague, Sir Thomas Smith. A violent quarrel took place between them while the negotiations were in progress, but the treaty of Troyes was finally signed on 1 April 1564, whereupon Throckmorton withdrew from the French embassy.

Next year another diplomatic mission was provided for Throckmorton in Scotland. On 4 May 1565 instructions were drawn up directing him to proceed to Scotland to prevent the marriage of Mary Queen of Scots with Darnley. He hurried to Mary at Stirling Castle. The queen received him reluctantly, and turned a deaf ear to his protest against her union with her cousin. He returned home leisurely, pausing at York to send Cecil the result of his observations on the temper of northern England, where he detected disquieting signs of hostility to Elizabeth's government. Later in the year he addressed a letter of advice to Mary urging her to show clemency to the banished protestant lords, and especially to the Earl of Moray (, Memoirs, 1683, pp. 60–3).

Throckmorton was created M.A. at Oxford on 2 Sept. 1566, and next year was, on the recommendation of the Earl of Leicester, named a governor of the incorporated society which was to control the possessions and revenues of the preachers of the gospel in Warwickshire. On 30 June 1567 Throckmorton was ordered to proceed to Scotland for a second time. A dangerous crisis had just taken place in Queen Mary's affairs. Her recent marriage to Bothwell after Darnley's murder had led to the rebellion of the Scottish nobles, and they had in June imprisoned her in Lochleven Castle. As a believer in the justice of Mary's claims to the English succession and an admirer of her personal charm, Throckmorton was anxious to alleviate the perils to which she was exposed. Elizabeth's instructions gave him no certain guidance as to the side on which he was to throw English influence. He travelled slowly northwards, in the hope that Elizabeth would adopt a clearer policy. On arriving at Edinburgh in July he told Mary at a personal interview that Queen Elizabeth would come to her rescue if she would abandon Bothwell. His persuasions were in vain (MS. Cotton, Calig. C. 1, ff. 18–35), but on 24 July the imprisoned queen wrote thanking him for the good feeling he had shown her (, Lettres, ii. 63). At the same time he opened negotiations with the Scottish lords. Elizabeth reproached him with his failure to secure Queen Mary's release (, Scottish State Papers, ii. 824–46). In self-defence Throckmorton disclosed to the Scottish lords his contradictory orders, but the queen resented so irregular a procedure, and he was recalled in August (cf., Memoirs, 96 seq.).

Throckmorton thenceforth suffered acutely from a sense of disappointment. His health failed during 1568, but he maintained friendly relations with Cecil, to whom he wrote from Fulham on 2 Sept. 1568 that he proposed to kill a buck at Cecil's house at Mortlake. He had long favoured the proposal to wed Queen Mary to the Duke of Norfolk, and he was consequently suspected next year of sympathy with the rebellion of northern catholics in Queen Mary's behalf. In September 1569 he was imprisoned in Windsor Castle, but he was soon released and no further proceedings were taken against him. He died in London on 12 Feb. 1570–1. Shortly before he had dined or supped with the Earl of Leicester at Leicester House. According to the doubtful authority of Leicester's ‘Commonwealth,’ his death was due to poison administered by Leicester in a salad on that occasion (, Commonwealth, 1641, p. 27). Leicester, it is said, had never forgiven Throckmorton for his vehement opposition to the earl's proposed marriage with the queen. No reliance need be placed on this report. Throckmorton had continuously corresponded on friendly terms with Leicester for many years before his death, and they had acted together as patrons of puritan ministers (cf., Scottish Papers, i. 210 seq.; Cal. State Papers, Dom. 1547–80, p. 291); Cecil wrote to Sir Thomas Smith of their markedly amicable relations on 16 Oct. 1565, and described Throckmorton as ‘carefull and devote to his lordship’ (, Life and Times of Elizabeth, i. 209). Throckmorton was buried on the south side of the chancel in St. Catherine Cree Church in the city of London.

Throckmorton married Anne, daughter of Sir Nicholas Carew, K.G., and sister and heiress of Sir Francis Carew of Beddington, Surrey. Of three daughters, Elizabeth (baptised at Beddington 16 April 1565) married Sir Walter Ralegh [q. v.] Of two sons, the elder, Arthur (1557–1626), matriculated from Magdalen College, Oxford, in 1571, aged 14; he was M.P. for Colchester in 1588–9; joined in 1596 the expedition to Cadiz, where he was knighted; inherited from his father the manor of Paulerspury, Northamptonshire, of which county he was sheriff in 1605, and was buried at Paulerspury on 1 Aug. 1616.