AbstractNitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca2+/CaM‐dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L‐arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane‐bound. Molecular cloning of the full‐length Stramonita NOS (Sh‐NOS) by reverse‐transcription polymerase chain reaction (RT‐PCR) followed by 5′ and 3′ RACE showed that Sh‐NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N‐terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh‐NOS gene was evaluated by RT‐PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods. J. Comp. Neurol. 520:364–383, 2012. © 2011 Wiley Periodicals, Inc.