Uptake of iron from various siderophores by a deltafet3deltafet4 strain of Saccharomyces cerevisiae was investigated. The catecholate enterobactin and the hydroxamate coprogen were taken up by the cells by passive diffusion, whereas the hydroxamates ferrioxamine B (FOB) and ferricrocin (FC) were taken up via a high-affinity energy-dependent mechanism. The kinetics of FOB and FC uptake showed reciprocal competitive inhibition. The transport was regulated by iron availability, but was independent of the Aft1p and Mac1p transcriptional activators. Mutants affected in the transport of FOB were isolated. The transport of FC was not impaired in these mutants. Functional complementation of one mutant allowed the identification of the SIT1 gene (Siderophore Iron Transport) encoding a putative permease belonging to the major facilitator superfamily. The Sit1 protein is probably a permease specific for the transport of ferrioxamine-type siderophores. The evidence suggests that the uptake of ferrichrome-type siderophores like FC involves other specific permease(s), although there seems to be a common handling of FOB and FC following their internalization by the cell.