Recent studies have provided evidence for a dual role of nitric oxide (NO) during murine leishmaniasis. To explore this problem, we monitored the formation of NO and its derived oxidants during the course of Leishmania amazonensis infection in tissues of susceptible (BALB/c) and relatively resistant (C57BL/6) mice. NO production was detected directly by low-temperature electron paramagnetic resonance spectra of animal tissues. Both mouse strains presented detectable levels of hemoglobin nitrosyl (HbNO) complexes and of heme nitrosyl and iron-dithiol-dinitrosyl complexes in the blood and footpad lesions, respectively. Estimation of the nitrosyl complex levels demonstrated that most of the NO is synthesized in the footpad lesions. In agreement, immunohistochemical analysis of the lesions demonstrated the presence of nitrotyrosine in proteins of macrophage vacuoles and parasites. Since macrophages lack myeloperoxidase, peroxynitrite is likely to be the nitrating NO metabolite produced during the infection. The levels of HbNO complexes in the blood reflected changes occurring during the infection such as those in parasite burden and lesion size. The maximum levels of HbNO complexes detected in the blood of susceptible mice were higher than those of C57BL/6 mice but occurred at late stages of infection and were accompanied by the presence of bacteria in the cutaneous lesions. The results indicate that the local production of NO is an important mechanism for the elimination of parasites if it occurs before the parasite burden becomes too high. From then on, elevated production of NO and derived oxidants aggravates the inflammatory process with the occurrence of a hypoxic environment that may favor secondary infections.