The aims of this study were to purify and localize the nitric oxide synthases (NOSs) from hybrid tilapia (Nile tilapia Oreochromis niloticus x Mozambique tilapia O. mossambicus). The purification procedures involved affinity chromatography with a 2', 5'-ADP-agarose 4B column and ion exchange with a diethylaminoethanol Bio-Gel A column. The results from gel filtration assays showed that the molecular weights of neuronal NOS (nNOS) and inducible NOS (iNOS) were 178 and 120 kDa, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that there were three bands with molecular weights of 89, 47, and 29 kDa from the purified nNOS. However, only one band, with a molecular weight of 120 kDa, appeared on the gel from the purified iNOS. Hybrid tilapia nNOS was a dimer structure, while iNOS appeared to be a monomer structure. Moreover, our results revealed that the activities of nNOS and iNOS were significantly higher after the addition of Ca+2 or Mg+2 ions individually. However, when L-arginine and NADPH were present, the addition of 1 mM of either ion did not further increase the activity. The chemical L-N(G)-methyl-L-arginine could inhibit the activities of the purified NOSs with or without L-arginine. Western blot analyses showed only an 89-kDa immunoreactive band from the extracts of cerebrum; however, we did not find the specific bands in other tissues, such as gill, intestine, liver, spleen, and anterior kidney. We found another 120-kDa immunoreactive protein band with the rabbit antirat iNOS serum against iNOS from the extracts of anterior kidney and spleen. The results of immunohistochemistry with the rabbit antihuman nNOS serum indicated that the nNOS existed in the cerebellum, olfactory bulb, diencephalons, and nerve cell bodies and neuronal fibers of the spinal cord. Interestingly, only macrophages from anterior kidney and spleen showed positive reactions with the rabbit antirat iNOS serum. In the same way, the endothelial NOS (eNOS) located in the heart and epithelial cells of the blood vessels reacted positively with the rabbit antibovine eNOS serum.