The Src family kinases (SFKs) play essential roles in collagen- and von Willebrand factor (VWF)-mediated platelet activation. However, the roles of SFKs in G protein-coupled receptor-mediated platelet activation and the molecular mechanisms whereby SFKs are activated by G protein-coupled receptor stimulation are not fully understood. Here we show that the thrombin receptor protease-activated receptor 4 agonist peptide AYPGKF elicited SFK phosphorylation in P2Y(12) deficient platelets but stimulated minimal SFK phosphorylation in platelets lacking G(q). We have previously shown that thrombin-induced SFK phosphorylation was inhibited by the calcium chelator 5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA). The calcium ionophore A23187 induced SFK phosphorylation in both wild-type and G(q) deficient platelets. Together, these results indicate that SFK phosphorylation in response to thrombin receptor stimulation is downstream from G(q)/Ca(2+) signaling. Moreover, A23187-induced thromboxane A(2) synthesis, platelet aggregation, and secretion were inhibited by preincubation of platelets with a selective SFK inhibitor, PP2. AYPGKF-induced thromboxane A(2) production in wild-type and P2Y(12) deficient platelets was abolished by PP2, and AYPGKF-mediated P-selectin expression, integrin α(IIb)β(3) activation, and aggregation of P2Y(12) deficient platelets were partially inhibited by the PKC inhibitor Ro-31-8220, PP2, dimethyl-BAPTA, or LY294002, but were abolished by Ro-31-8220 plus PP2, dimethyl-BAPTA, or LY294002. These data indicate that Ca(2+)/SFKs/PI3K and PKC represent two alternative signaling pathways mediating G(q)-dependent platelet activation.