1 In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide snythase was determined biochemically and immunochemically. 2 In the rat brain, the highest nitric oxide snythase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by N(G)-nitro-L-arginine or N(G)-methyl-L-arginine. 3 Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4 These results indicate that the ferret brain contains a nitric oxide snythase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain.