Afonso de Albuquerque was born in 1453 in Alhandra, near Lisbon. He was the second son of Goncalo de Albuquerque, Lord of Vila Verde dos Francos, and Dona Leonor de Menezes. His father held an important position at court and was connected by remote illegitimate descent with the Portuguese monarchy. He was educated in mathematics and Latin at the court of Afonso V of Portugal, where he befriended Prince John, the future King John II of Portugal.

Afonso arrived at Cannanore on the Malabar coast in December 1508, where he opened before the viceroy, Dom Francisco de Almeida, the sealed letter which he had received from the King, and which named as governor to succeed Almeida. The viceroy, supported by the officers who had abandoned Afonso at Ormuz, had a matching royal order, but declined to yield, protesting that his term ended only in January and stating his intention to avenge his son's death by fighting the Mamluk fleet of Mirocem, refusing Afonso's offer to fight him himself. Afonso avoided confrontation, which could have led to civil war, and moved to Kochi, India, to await further instruction from the King, maintaining his entourage himself. He was described by Fernao Lopes de Castanheda as patiently enduring open opposition from the group that had gathered around Almeida, with whom he kept formal contact. Increasingly isolated, he wrote to Diogo Lopes de Sequeira, who arrived in India with a new fleet, but was ignored as Sequeira joined the Viceroy. At the same time, Afonso refused approaches from opponents of the Viceroy, who encouraged him to seize power.  On 3 February 1509, Almeida fought the naval Battle of Diu against a joint fleet of Mamluks, Ottomans, the Zamorin of Calicut, and the Sultan of Gujarat, regarding it as personal revenge for the death of his son. His victory was decisive: the Ottomans and Mamluks abandoned the Indian Ocean, easing the way for Portuguese rule there for the next century. In August, after a petition from Afonso's former officers with the support of Diogo Lopes de Sequeira claiming him unfit for governance, Afonso was sent in custody to St. Angelo Fort in Cannanore. There he remained under what he considered to be imprisonment.  In September 1509, Sequeira tried to establish contact with the Sultan of Malacca but failed, leaving behind 19 Portuguese prisoners.

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