In washed platelet systems, thrombin has been demonstrated to downregulate the platelet surface expression of glycoprotein (GP) Ib and GPIX. In the present study, we addressed the question as to whether, in the more physiologic milieu of whole blood, downregulation of platelet surface GPIb and GPIX can be induced by thrombin, adenosine diphosphate (ADP), and/or by an in vivo wound. Thrombin-induced downregulation of GPIb and GPIX on the surface of individual platelets in whole blood was demonstrated by the use of flow cytometry, a panel of monoclonal antibodies (MoAbs) and, to inhibit fibrin polymerization, the peptide glycyl-L-prolyl-L-arginyl-L-proline. Platelets were identified in whole blood by a GPIV-specific MoAb and exclusion of monocytes by light scattering properties. Flow cytometric analysis of whole blood emerging from a standardized bleeding-time wound established that downregulation of platelet surface GPIb and GPIX can occur in vivo. A GPIb-IX complex-specific antibody indicated that the GPIb and GPIX remaining on the surface of platelets activated in vivo or in vitro were fully complexed. Simultaneous analysis of individual platelets by two fluorophores demonstrated that thrombin-induced platelet surface exposure of GMP-140 (degranulation) was nearly complete at the time that downregulation of platelet surface GPIb-IX was initiated. However, degranulation was not a prerequisite because ADP downregulated platelet surface GPIb-IX without exposing GMP-140 on the platelet surface. Inhibitory effects of cytochalasins demonstrated that the activation-induced downregulation of both GPIX and GPIb are dependent on actin polymerization. In summary, downregulation of the platelet surface GPIb-IX complex occurs in whole blood stimulated by thrombin, ADP, or an in vivo wound, and is independent of alpha granule secretion.