Sodium nitroprusside (SNP), a generator of nitric oxide (NO), decreased [3H]dopamine uptake into rat striatal synaptosomal preparations in a dose-, time- and temperature-dependent fashion. Reduced hemoglobin, a substance that binds NO, prevented the SNP-induced decrease in uptake. Potassium ferri- and ferrocyanide, compounds similar to SNP that do not produce NO, were without effect on uptake. SNP inhibited [3H]dopamine uptake in synaptosomes from nucleus accumbens and olfactory tubercle as well but with a lower potency. SNP inhibited [3H]serotonin and [3H]glutamate uptake but had no effect on [3H]norepinephrine uptake. S-Nitroso-N-acetylpenicillamine (SNAP), another generator of NO, had effects similar to those of SNP. The SNP-induced decrease in [3H]dopamine uptake was due to a Vmax decrease at 100 microM SNP and to both a Vmax and Km change at 300 microM SNP. Depletion of calcium by omission of calcium from buffers and addition of EGTA increased the potency of SNP in inhibiting uptake. There was no change in [3H]WIN 35428 binding to the dopamine transporter with doses of SNP that inhibited uptake. These data indicate that NO can decrease [3H]dopamine transporter function.