Rap1, a small GTPase of the Ras family, is ubiquitously expressed and particularly abundant in platelets. Previously we have shown that Rap1 is rapidly activated after stimulation of human platelets with α-thrombin. For this activation, a phospholipase C-mediated increase in intracellular calcium is necessary and sufficient. Here we show that thrombin induces a second phase of Rap1 activation, which is mediated by protein kinase C (PKC). Indeed, the PKC activator phorbol 12-myristate 13-acetate induced Rap1 activation, whereas the PKC-inhibitor bisindolylmaleimide inhibited the second, but not the first, phase of Rap1 activation. Activation of the integrin α_IIbβ₃, a downstream target of PKC, with monoclonal antibody LIBS-6 also induced Rap1 activation. However, studies with α_IIbβ₃-deficient platelets from patients with Glanzmann's thrombasthenia type 1 show that α_IIbβ₃ is not essential for Rap1 activation. Interestingly, induction of platelet aggregation by thrombin resulted in the inhibition of Rap1 activation. This downregulation correlated with the translocation of Rap1 to the Triton X-100-insoluble, cytoskeletal fraction. We conclude that in platelets, α-thrombin induces Rap1 activation first by a calcium-mediated pathway independently of PKC and then by a second activation phase mediated by PKC and, in part, integrin α_IIbβ₃. Inactivation of Rap1 is mediated by an aggregation-dependent process that correlates with the translocation of Rap1 to the cytoskeletal fraction.