In experimental visceral leishmaniasis, in which the tissue macrophage is the target, in vivo responsiveness to conventional chemotherapy (pentavalent antimony [Sb]) requires a T-cell-dependent mechanism. To determine if this mechanism involves gamma interferon (IFN-γ)-induced activation and/or specific IFN-γ-regulated macrophage leishmanicidal mechanisms (generation of reactive nitrogen or oxygen intermediates, we treated gene-deficient mice infected withLeishmania donovani. In IFN-γ gene knockout (GKO) mice, Sb inhibited but did not kill intracellular L. donovani(2% killing versus 76% in controls). Sb was active (>94% killing), however, in both inducible nitric oxide synthase (iNOS) knockout (KO) and respiratory burst (phagocyte oxidase)-deficient chronic granulomatous disease (X-CGD) mice. Sb's efficacy was also maintained in doubly deficient animals (X-CGD mice treated with an iNOS inhibitor). In contrast to Sb, amphotericin B (AmB) induced high-level killing in GKO mice; AmB was also fully active in iNOS KO and X-CGD animals. Although resolution of L. donovani infection requires iNOS, residual visceral infection remained largely suppressed in iNOS KO mice treated with Sb or AmB. These results indicate that endogenous IFN-γ regulates the leishmanicidal response to Sb and achieves this effect via a pathway unrelated to the macrophage's primary microbicidal mechanisms. The role of IFN-γ is selective, since it is not a cofactor in the response to AmB. Treatment with either Sb or AmB permits an iNOS-independent mechanism to emerge and control residual intracellular L. donovani infection.