Background. Although Leishmania virulence may be modulated by environmental and genetic factors of their mammalian hosts and sand fly vectors, molecular determinants of Leishmania sp. are the key elements. This work evidences that Leishmania amazonensis axenic amastigotes produce comparatively more NO than infective promastigotes. Methods. A soluble NOS was purified from L. amazonensis axenic amastigotes by affinity chromatography (2',5'-ADP-agarose), and on SDS-PAGE the enzyme migrates as a single protein band. Results. The presence of a constitutive NOS was detected through immunofluorescence using antibody against neuronal NOS (nNOS) and in NADPH consumption assays. Conclusions. The present data show that NOS is prominent in axenic amastigote preparations, suggesting an association with the infectivity and/or an escaping mechanism of the parasite. The relationship between the NO-generating systems in the parasite and in their host cell warrants further investigation.