Mutational analysis has established that the cytoplasmic tail of the integrin beta 3 subunit binds c-Src (termed as Src in this study) and is critical for bidirectional integrin signaling. Here we show in washed human platelets that a cell-permeable, myristoylated RGT peptide (myr-RGT) corresponding to the integrin beta 3 C-terminal sequence dose-dependently inhibited stable platelet adhesion and spreading on immobilized fibrinogen, and fibrin clot retraction as well. Myr-RGT also inhibited the aggregation-dependent platelet secretion and secretion-dependent second wave of platelet aggregation induced by adenosine diphosphate, ristocetin, or thrombin. Thus, myr-RGT inhibited integrin outside-in signaling. In contrast, myr-RGT had no inhibitory effect on adenosine diphosphate-induced soluble fibrinogen binding to platelets that is dependent on integrin inside-out signaling. Furthermore, the RGT peptide induced dissociation of Src from integrin beta 3 and dose-dependently inhibited the purified recombinant beta 3 cytoplasmic domain binding to Src-SH3. In addition, phosphorylation of the beta 3 cytoplasmic tyrosines, Y(747) and Y(759), was inhibited by myr-RGT. These data indicate an important role for beta 3-Src interaction in outside-in signaling. Thus, in intact human platelets, disruption of the association of Src with beta 3 and selective blockade of integrin alpha IIb beta 3 outside-in signaling by myr-RGT suggest a potential new antithrombotic strategy.