AbstractThe experiments described in this report were aimed at determining whether L‐arginine (L‐arg)‐derived nitrogen oxidation products (nitric oxide, nitrous acid, nitrites) are involved in the intracellular killing ofLeishmaniaparasites by activated murine macrophages in vitro. Peritoneal or bone marrow‐derived macrophages were infected withL. enriettiiorL. major, then activated by exposure to recombinant murine interferon‐gamma or to macrophage activating factor (MAF)‐rich media in the presence of li‐popolysaccharide. Activation of macrophages in regular (i.e., arginine‐containing) culture medium led to complete destruction of the microorganisms within 24 h (L. enriettii)or 48 h (L.major), concomitant with accumulation of nitrites (NO2‐) in the culture fluids. When macrophage activation was carried out in L‐arg‐free medium, however, neither parasite killing nor NO2‐production was obtained. A similar inhibition of macrophage leishmanicidal activity and of NO2‐release was observed using media treated with arginase (which converts L‐arg to urea and ornithine), or supplemented with NG‐monomethyl‐L‐arg or guanidine (which inhibit the conversion of L‐arg to nitrogen oxidation products). In all these situations, an excellent correlation between the levels of NO2‐production by macrophages and intracellular killing ofLeishmaniawas observed, whereas no strict correlation was detectable between leishmanicidal activity and superoxide production. Intracellular parasite killing by activated macrophages could be prevented by addition of iron salts to the incubation fluids. Incubation of free parasites with NaNO2at acid pH (which permits the production of nitrous acid) led to immobilisation, multiplication arrest, and morphological degeneration of the microorganisms. Similarly, exposure of infected cells to NaNO2led to killing of the intracellular parasite without affecting macrophage viability. These experiments strongly suggest that the leishmanicidal effect of activated murine macrophages involves the agency of L‐arg‐derived nitrogen oxidation products.