We investigated the role of intracellular iron on the capacity of Histoplasma capsulatum (Hc) yeasts to multiply within human macrophages (Mphi). Coculture of Hc-infected Mphi with the iron chelator deferoxamine suppressed the growth of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by iron-free transferrin (apotransferrin). Chloroquine, which prevents release of iron from transferrin by raising endocytic and lysosomal pH, induced human Mphi to kill Hc. The effect of chloroquine was reversed by iron nitriloacetate, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. Chloroquine (40-120 mg/kg) given intraperitoneally for 6 d to Hc-infected C57BL/6 mice significantly reduced the growth of Hc in a dose-dependent manner. At 120 mg/kg there was a 17- and 15-fold reduction (P < 0.01) in CFU in spleens and livers, respectively. The therapeutic effect of chloroquine also correlated with the length of treatment. As little as 2 d of chloroquine therapy (120 mg/kg), when started at day 5 after infection, reduced CFU in the spleen by 50%. Treatment with chloroquine for 10 d after a lethal inoculum of Hc protected six of nine mice; all control mice were dead by day 11 (P = 0.009). This study demonstrates that: (a) iron is of critical importance to the survival and multiplication of Hc yeasts in human Mphi; (b) in vitro, chloroquine induces Mphi killing of Hc yeasts by restricting the availability of intracellular iron; and (c) in vivo, chloroquine significantly reduces the number of organisms in the spleens and livers of Hc-infected mice and can protect mice from a lethal inoculum of Hc yeasts. Thus, chloroquine may be effective in the treatment of active histoplasmosis and also may be useful in preventing relapse of histoplasmosis in patients with acquired immunodeficiency syndromes.