The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).