The characteristics of the binding [3H]6-nitroquipazine, a very potent and selective inhibitor of 5-hydroxytryptamine (5-HT; serotonin) uptake, to human platelet membranes were studied at a physiological temperature of 37 degrees C. The presence of a single saturable high-affinity binding component for [3H]6-nitroquipazine was demonstrated Non-specific binding was estimated in the presence of 1 microM paroxetine. Scatchard analysis revealed an apparent equilibrium dissociation constant (Kd) of 0.450 +/- 0.04 nM and a maximal number of binding sites (Bmax) of 2508 +/- 360 fmol/mg protein (mean +/- S.D., n = 4). The kinetically derived dissociation constant (Kd) was 0.431 nM. [3H]6-Nitroquipazine binding was inhibited selectively by 5-HT uptake inhibitors, and the potency of various drugs to inhibit [3H]6-nitroquipazine binding closely correlated with their inhibitory effects on [3H]5-HT uptake into synaptosome. Moreover, Ki values for drug inhibition of [3H]6-nitroquipazine binding to human platelet membranes were significantly correlated with the corresponding Ki values for inhibition of [3H]paroxetine binding at 37 degrees C. The present results suggest that the binding sites for [3H]6-nitroquipazine are associated with the 5-HT transporter in human platelets.