We studied the effects of intracerebroventricular and intraperitoneal injection and the in vitro effects of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, on the nitric oxide synthase activities of the cerebellum, brainstem, hypothalamus, hippocampus, and the remainder of the brain after dissections. Male rats were chronically implanted with lateral icv guide cannula. L-NAME was injected in doses of 0.2, 1, and 5 mg intracerebroventricularly, and 50 mg/kg intraperitoneally. L-NAME induced dose-dependent suppression of NOS activities in each brain region. The threshold dose was 0.2 mg; 1 mg L-NAME completely abolished brain nitric oxide synthase activity 90 min after the injection. Brain NOS activities returned to baseline level 48 h after the injection of 5 mg L-NAME. There were significant differences between the sensitivity of various regions to L-NAME after in vivo but not in vitro administration of the enzyme inhibitor. These findings indicate that intracerebroventricular injection of L-NAME is a useful tool for inhibiting brain nitric oxide synthase activities in vivo. The differences between the sensitivity of different brain regions to L-NAME as well as the relative fast recovery of nitric oxide synthase activities must be taken into account when L-NAME is administered intracerebroventricularly to rats.