Binding of human [125I]thrombin to washed human platelets was studied in order to analyze the nonenzymic aspects of the thrombin stimulation of platelets. Highly purified alpha-thrombin that was iodinated with lactoperoxidase retained full clotting and esterase activities and full activity toward platelets, was not distinguished from native thrombin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel chromatography, and bound to platelets the same as unlabeled thrombin. Bound and free [125I]thrombin were measured after rapid separation of platelets from the suspending medium by centrifugation through oil. Maximum binding was within 15 s, the shortest time measured. At concentrations of thrombin sufficient to cause less than maximal platelet stimulation, 90% of the total thrombin was free in the suspending solutions. Equilibrium binding was established, with both free thrombin and free platelets retaining activity, and with rapid reequilibration after dilution or addition of unlabeled thrombin. The equilibrium was complex, with the apparent number of binding sites and dissociation constants dependent on thrombin concentration. Analysis of bound thrombin as a function of thrombin concentration by double-reciprocal and Scatchard plots indicated 300-400 high affinity sites (Kdiss = 1.8-2 nM); these correlate with thrombin stimulation of Ca2+ secretion, which shows half maximal effect at 1.5 nM thrombin and maximal effect with 500-600 thrombins bound per platelet.