Macrophages infected with amastigotes of Leishmania major and treated with IFN-gamma in vitro develop potent antimicrobial activities that eliminate the intracellular parasite. This antileishmanial activity was suppressed in a dose dependent fashion by NG-monomethyl-L-arginine (NGMMLA), a competitive inhibitor of nitrite, nitrate, nitric oxide and L-citrulline synthesis from L-arginine. Excess L-arginine added to infected macrophage cultures reversed the inhibitory effects of NGMMLA. Addition of arginase to culture media inhibited intracellular killing by IFN-gamma-treated cells. Similar effects were seen with macrophages obtained from BCG-infected C3H/HeN mice. Increased levels of nitrite, an oxidative product of the L-arginine-dependent effector mechanism, was measured in cultures of infected IFN gamma-treated macrophages as well as infected BCG-activated macrophages. Nitrite production correlated with development of antileishmanial activity. Nitrite production and microbicidal activity both decreased when in vivo or in vitro-activated macrophages were cultured in the presence of either arginase or NGMMLA. Nitric oxide synthesized from a terminal guanidino nitrogen atom of L-arginine and a precursor of the nitrite measured, may disrupt Fe-dependent enzymatic pathways vital to the survival of amastigotes within macrophages.