Binding of prothrombin and activation intermediates 1 and 2 to human platelets was tested with 125-I-labeled protein preparations. None of these precursors of thrombin bound to platelets under conditions in which high affinity binding of thrombin was observed,nor did they cause platelet aggregation or serotonin release. The molecular conformation required for binding to platelets as well as for induction of platelet aggregation and release is present, therefore, only after the final step in prothrombin activation.