The distribution and transport of thrombospondin (TSP), fibrinogen (Fbg), fibronectin (Fn), and Factor VIII-related antigen (VIII:RAg) in resting and thrombin-stimulated platelets was investigated by immunofluorescence microscopy. In resting intact cells, little surface staining was seen for these proteins. In permeable resting cells, punctate staining similar to that reported for platelet factor 4 was observed. Double-label immunofluorescence staining for Fbg and either beta-thromboglobulin (beta TG), TSP, or Fn demonstrated co-localization, indicating their presence in the same intracellular structures. VIII:RAg showed general co-localization; however, the staining was finer, suggesting a possible differential intragranular localization. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins. In thrombin-stimulated cells, co-localization of all proteins in these masses was observed by double label immunofluorescence. Thus, TSP, Fbg, Fn, and beta TG are localized in the same structure in resting cells. Thrombin stimulates formation of common larger masses of these proteins prior to their release, suggesting that they reach the cell surface through a common intermediate.