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De Ayllón's expedition sailed from Hispaniola to South Carolina on two ships; the settlers included African slaves. The colony, called San Miguel de Guadalupe, was disrupted by a fight over leadership. During this turmoil, the slaves revolted and fled the colony to live among the Native Americans (the Cofitachiqui). The colony soon came to an end as de Ayllón and the other colonists died in a fever epidemic.
Before this disastrous venture at colonization, de Ayllón had sailed to the West Indies from Spain in 1502 to be a judge in Hispaniola (Santo Domingo). He later mediated the dispute between Hernán Cortés and Diego Velázquez in Mexico (1520).
De Ayllon was later sent to the coast of South Carolina by the Holy Roman emperor Charles V (King Charles I of Spain) to search for a water route to the Spice Islands (in the Pacific Ocean). Although he failed at this impossible task, De Ayllón landed near Cape Fear in 1523 and explored the area.
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