[3H]Serotonin (5-HT) uptake by synaptosomes of rat brain was dose-dependently inhibited by nitric oxide (NO) donors such as sodium nitroprusside (SNP), 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamin e, 3-morpholinosydnonimine and S-nitroso-L-cysteine (NO-CYS). The inhibitory effect was blocked by reduced hemoglobin. The effect was not mimicked by ferrocyanide and ferricyanide. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-bromo cGMP) did not affect [3H]5-HT uptake into rat cortical synaptosomes. The reduced activity of [3H]5-HT uptake into the cortical synaptosomes pretreated with NO-CYS was partially reversed by washing the preparation after the treatment. Kinetic analysis showed that NO-CYS (100 microM) decreased the Vmax value without any change in the Km value. NO-CYS did not affect the specific binding of [3H]paroxetine, a ligand that binds to the 5-HT transporter, in membranes. NO-CYS and SNP, like iodoacetic acid and sodium cyanide, decreased the ATP content in cortical synaptosomes, but the effect on ATP content was not related to that on [3H]5-HT uptake. These findings suggest that NO inhibits reversibly [3H]5-HT uptake into rat brain synaptosomes without affecting the recognition site of the 5-HT transporter in a cGMP-independent manner, and the observed effect is not due to its metabolic effect.