Binding of 125I-labeled thrombin to isolated human platelet plasma membranes was studied. Two classes of sites, one with high and one with low affinity for thrombin, were demonstrated. The apparent dissociation constants for the high and low affinity sites were 3.2 and 600 nM, respectively, similar to values obtained with intact platelets. Maximum binding was within 10 s, the shortest time measured, and then decreased with time to a constant level of binding within 45 s. When the equilibrium was perturbed by dilution, the system re-equilibrated with less thrombin bound than in a control that was dilute before mixing thrombin and membranes. Neither the time-dependent decrease nor the dilution effect were observed with phenylmethylsulfonyl-125I-labelled thrombin, an irreversibly inhibited thrombin, suggesting that these phenomena may involve a thrombin-catalyzed modification of the membranes leading decreased binding.