Nitric oxide (NO) derived from activated macrophages has been shown to be crucial for the host's leishmanicidal activities. Excess NO, however, can induce apoptosis in some cell types, including macrophages. In the present investigation, we studied the role of NO in inducing apoptosis of BALB/c mice macrophages infected with Leishmania major in vitro. The peritoneal macrophages were harvested and cultured with or without L.major in the presence of a donating reagent (s-Nitroso-N-Acetylpenicillamine (SNAP)) or an inhibitor of NO synthase (NG -Methyl-L-Arginine (NMMA)). The concentration of NO in culture supernatants was measured after 18 hours incubation. Simultaneously, macrophages undergoing apoptosis were identified by fluorescence and electron microscopy. The results showed an increase in apoptosis rate in parallel to nitrite production in macrophages cultured in the presence of SNAP. Although macrophages infected with L.major had no significant increase in NO production, they showed a significant increase in apoptosis rate. Besides, macrophages cultured with NMMA, had a decreased NO production but the apoptosis rate increased. Therefore, mechanisms involved in apoptosis induction in the last two groups may be different from NO overproduction.