A rarely occurring variant of human serotonin transporter (hSERT) was tested for its functional consequences in HeLa and COS-7 cells. The variant, in which Ile-425 is converted to Val, was significantly different from wild type with respect to its catalytic properties. In both cell types, rates of serotonin (5-HT) transport were higher for the I425V variant. Both an increase in Vmax and a decrease in KM caused this increase in rate. The increase in Vmax was not accounted for by increases in transporter expression or in the distribution of transporter between the cell surface and intracellular pools. The decrease in KM was accompanied by a decrease in the KD for binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane. In both HeLa and COS-7 cells, the nitric oxide donor S-nitroso-N-acetylpenicillamine increased the activity of wild-type hSERT to that of the variant but did not change the activity of the I425V variant. This stimulation was prevented by the presence of oxyhemoglobin, which quenches nitric oxide, and by an inhibitor of guanylyl cyclase.