The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.