Leishmania chagasi, the cause of South American visceral leishmaniasis, requires iron for its growth. However, the extent to which different iron sources can be utilized by the parasite is not known. To address this question, we studied acquisition of iron from lactoferrin and transferrin by the extracellular promastigote form of L. chagasi during growth in vitro. A promastigote growth medium based on minimal essential medium supplemented with iron-depleted serum supported promastigote growth only after the addition of exogenous iron. The addition of 8 microM iron chelated to lactoferrin or hemin resulted in normal promastigote growth. Ferritransferrin also supported promastigote growth, but only after a considerable lag. Promastigotes grown in all three iron sources generated similar amounts of hydroxyl radical upon exposure to hydrogen peroxide, indicating that none of these protected parasites against generation of this toxic radical. Promastigotes were able to take up 59Fe chelated to either transferrin or lactoferrin, although uptake from 59Fe-lactoferrin occurred more rapidly. 59Fe uptake from either 59Fe-transferrin or 59Fe-lactoferrin was inhibited by a 10-fold excess of unlabeled ferrilactoferrin, ferritransferrin, apolactoferrin, apotransferrin, or iron nitrilotriacetate but not ferritin or bovine serum albumin. There was no evidence for a role for parasite-derived siderophores or proteolytic cleavage of ferritransferrin or ferrilactoferrin in the acquisition of iron by promastigotes. Thus, L. chagasi promastigotes can acquire iron from hemin, ferrilactoferrin, or ferritransferrin. This capacity to utilize several iron sources may contribute to the organism's ability to survive in the diverse environments it encounters in the insect and mammalian hosts.