The antimicrobial effect of activated macrophages on parasites involves nitric oxide (NO). NO induces intracellular parasite killing in murine leishmaniasis. Nevertheless, the mechanisms of action of NO as a final effector molecule on intracellular forms of Leishmania are unknown. The recent development of axenically grown amastigote forms of different Leishmania species allowed direct investigation of NO activity on active and dividing populations of the mammalian stage of various Leishmania species, which normally are only found intracellularly. Authentic NO gas, which reproduced the antimicrobial effect elaborated by activated macrophages, was flushed on promastigote and axenically cultured amastigote forms of L. mexicana, L. amazonensis, and L. chagasi suspended in degassed phosphate-buffered saline (PBS). After NO treatment, the viability of parasites gradually decreased as a function of time postflushing when compared to controls. Interestingly NO killing was more effective on promastigote forms than on amastigote forms. After 12-hr postflushing incubation in PBS, cultures of NO-treated parasites, contrary to controls (N2-treated), failed to proliferate whatever the species and the developmental stage considered. Addition of both FeSO4 and L-cysteine to PBS immediately after NO treatment reversed the capacity of authentic NO gas to inhibit the multiplication of both parasite stages of Leishmania. Supplementation of PBS with alpha-ketoglutarate and cis-aconitate (citric acid cycle substrates) also reversed the leishmanicidal activity of NO, whereas addition of citrate was less effective. The course of the developmental life cycle in vitro was also inhibited by NO gas treatment. Enzymatic analysis showed that aconitase activity was dramatically reduced by NO gas, whereas glucose phosphate isomerase, aspartate transferase, and phosphoglucomutase activities were unchanged. In accordance, promastigote and amastigote forms of Leishmania were shown to be killed by antimycin A, an inhibitor of mitrochondrial respiration. All these data demonstrated that NO action led to lethal metabolic inhibition in both developmental parasite stages by, at least in part, triggering iron loss from enzyme(s) with iron-sulfur prosthetic groups, in particular aconitase.