Normal human blood platelets in plasma were incubated at 2 degrees C with tritiated 5-hydroxytryptamine ([3H]5HT), and the specific receptor binding was displaced by the addition of unlabelled 5HT. The kinetic parameters of this binding were established and a two-site model for the platelet 5HT receptor demonstrated. Site A has a KD of 0.5-1 nM and capacity of 6-10 fmol/10(8) platelets, and site B a KD of 15-36 nM and a capacity of 100-150 fmol/10(8) platelets. Non-specific or non-receptor binding of [3H]5HT to platelets at 2 degrees C was resolved into a passive linear component and an active saturable component sensitive to metabolic inhibition. Binding to the lower affinity 5HT receptor site was inhibited by drugs of the tricyclic antidepressant type with IC50 values similar to those against the active uptake component of non-specific binding as described. Isomers of the neuroleptic drug flupenthixol showed a differential and competitive antagonism of high affinity [3H]5HT binding. The 5HT antagonist methysergide, and pizotifen and mianserin also were competitive inhibitors at this site. The rank order of potency of these drugs correlated with their action as inhibitors of 5HT induced platelet aggregation. It is concluded that binding of [3H]5HT to intact human platelets satisfies all the criteria for specific binding and that the two sites demonstrated, of high and lower affinity, are concerned with the functions of 5HT induced aggregation and 5HT uptake respectively.