The modified two-site model for platelet activation by collagen requires tight binding of platelets to collagen through integrin α2β1, after its prior activation by inside-out signals initiated by GP VI. The inside-out signalling to α2β1 is not well characterized although it is currently accepted that GPVI initiates signals that lead to regulation of this integrin. The aim of the study was to determine the role played by actin polymerization and the Rho family GTPase cdc42 in the regulation of α2β1 integrin. We first show that GPVI- and non-GPVI-dependent signals differentially regulate distribution of α2β1 receptors, where binding of platelets to collagen leads to redistribution of the integrin to areas of contact between platelet and collagen fibre. Binding of platelets to collagen also leads to activation of α2β1 integrin, which is dependent upon actin polymerization and cdc42 activity, since activation is blocked by cytochalasin D and secramine A respectively. Adhesion of platelets to collagen is markedly diminished in the presence of these inhibitors, whereas adhesion to CRP- or fibrinogen-coated surfaces is not affected. Platelet aggregation to collagen, but not CRP or thrombin, is also markedly dependent upon actin polymerization and cdc42 activity. In conclusion these data suggest that actin polymerization and cdc42 are required for activation of integrin α2β1, but not αIIbβ3, thereby critically regulating platelet adhesion to and activation by collagen. We therefore suggest a further modification to the current two-site two-step model for activation of platelets by collagen, where actin polymerization and cdc42 mediate a critical step in modulating α2β1 activation, possibly through a positive feedback pathway from α2β1 itself.