Previous studies suggest that nitric oxide (NO) may play a role in sleep regulation, particularly in the homeostatic process. The present studies were undertaken to compare the sleep effects of injecting a NO synthase (NOS) inhibitor when homeostatic sleep pressure is naturally highest (light onset) or when it is at its nadir (dark onset), in rats. Sleep, electroencephalogram (EEG) delta-wave activity during non-rapid-eye-movement sleep (NREMS), also known as slow-wave activity (SWA), and brain temperature responses to three doses of the NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME), (5, 50 and 100 mg/kg) injected intraperitoneally at light or dark onset were examined in rats (n = 6 to 8). The effects of 5 mg/kg L-NAME were determined in both normal and vagotomized (VX) rats. Light onset administration of 50 mg/kg L-NAME decreased NREMS amounts and suppressed SWA and increased rapid-eye-movement sleep (REMS) amounts. At dark onset, L-NAME injection also dose-dependently suppressed SWA, however, unlike light onset injections, both NREMS and REMS amounts were increased after all three doses. Sleep responses to 5 mg/kg L-NAME were not different in control and VX rats, suggesting that the sleep effects of L-NAME are not mediated through the activation of sensory vagal mechanisms. The present findings suggest that timing of the injection is a major determinant of the sleep responses observed after systemic L-NAME injection, in rats.