The presence, characteristics and localization of nitric oxide synthase (NOS) in the eyestalk ganglia of the crayfish (Procambarus clarkii) were investigated. Biochemical assay indicated that NOS activity was significantly inhibited by NG-nitro-L-arginine (a NOS inhibitor), fluphenazine-N-2-chloroethane (a calmodulin inhibitor) as well as by omitting β-NADPH or Ca2+ from the incubations. Immunohistochemical studies localized NOS immunoreactivity (IR) to the first chiasma and the sinus gland. Finally, a NOS-immunoreactive protein (138 kDa) was detected by Western blot analysis. These data demonstrate for the first time the existence in crustacean eyestalk ganglia of NOS with biochemical characteristics similar to mammalian constitutive NOS. The localization of NOS-IR suggests that NO may be involved in regulating visual processes and neuroendocrine function in crustaceans.