Leishmania is an obligate intracellular parasite that primarily inhabits macrophages. The destruction of the parasite in the host cell is a fundamental mechanism for infection control. In addition, inhibition of the leishmanicidal activity of macrophages seems to be related to the ability of some species to inhibit the production of nitric oxide (NO) by depleting arginine. Some species of Leishmania have the ability to produce NO from a constitutive nitric oxide synthase-like enzyme (cNOS-like). However, the localization of cNOS-like in Leishmania has not been described before. As such, this study was designed to locate cNOS-like enzyme and NO production in promastigotes of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. NO production was initially quantified by flow cytometry, which indicated a significant difference in NO production between L. (L.) amazonensis (GMFC = 92.17 +/- 4.6) and L. (V.) braziliensis (GMFC = 18.89 +/- 2.29) (P < 0.05). Analysis of cNOS expression by immunoblotting showed more expression in L. (L.) amazonensis versus L. (V.) braziliensis. Subsequently, cNOS-like immunolabeling was observed in promastigotes in regions near vesicles, the flagellar pocket and mitochondria, and small clusters of particles appeared to be fusing with vesicles suggestive of glycosomes, peroxisome-like-organelles that compartmentalize the glycolytic pathway in trypanosomatid parasites. In addition, confocal microscopy analysis demonstrated colocalization of cNOS-like and GAPDH, a specific marker for glycosomes. Thus, L. (L.) amazonensis produces greater amounts of NO than L. (V.) braziliensis, and both species present the cNOS-like enzyme inside glycosomes.