We have found conclusive evidence for inducible nitric oxide synthase (iNOS) activity in rainbow trout (Oncorhynchus mykiss) tissue by means of biochemical, immunohistochemical, and immunoblotting analyses. This Ca²⁺-independent enzyme uses L-arginine to produce nitric oxide and L-citrulline. It was significantly inhibited by the L-arginine analogs N-monomethyl-L-arginine and N^G-nitro-L-arginine methyl ester. Kinetic analyses showed typical Michaelian behavior with no evidence of cooperative effects. The specific activities of the liver and head kidney enzymes were 27 and 106 pmoles · min¹ · mg protein¹, respectively, with similar values for K_m (11 µM), all of which correspond well with the values for other previously characterized iNOS. Western blot analyses revealed a single band of M_R = 130 kDa tested with an iNOS antiserum. At the ultrastructural level, cells with NADPH-diaphorase activity and iNOS immunoreactivity were identified as being heterophilic granulocytes in head kidney tissue and neutrophils and macrophages in hepatic tissue. The presence of an iNOS isoform in these fish tissues implies that these cells are capable of generating nitric oxide, thus pointing to the potential role of this enzyme in fish defense mechanisms.