Plasma membranes of pig platelets were isolated and subjected to solubilization with digitonin. Binding of [3H]ketanserin to the solubilized membrane fraction, optimal at pH 7.5, was time and protein dependent and was greatly reduced by increasing ionic strength of the incubation buffer. Saturation analysis and inhibition of binding with various 5-HT2 antagonists showed the presence of two binding sites with high and low affinity, respectively. High affinity binding to solubilized membrane proteins correlated well with that found in native membranes with regard to the dissociation constant KD (1.6 and 1.2 nM, respectively) and the displacement by 5-HT2 antagonists. Binding to this site was not inhibited by the dopamine antagonist haloperidol which, however, displaced [3H]ketanserin bound to the low affinity site. At present the nature of the low affinity binding site (KD = 71 nM) is not well understood. Centrifugation on sucrose gradients revealed only one peak of [3H]ketanserin binding corresponding to the molecular weight of catalase (232,000 Da), thus probably reflecting a close association of the two binding sites.