Cofilin, in its Ser3 dephosphorylated form, accelerates actin filament turnover in cells. We report here the role of cofilin in platelet actin assembly. Cofilin is primarily phosphorylated in the resting platelet as evidenced by a specific antibody directed against its Ser3 phosphorylated form. After stimulation with thrombin under nonstirring conditions, cofilin is reversibly dephosphorylated and transiently incorporates into the actin cytoskeleton. Its dephosphorylation is maximal 1-2 min after platelet stimulation, shortly after the peak of actin assembly occurs. Cofilin rephosphorylation begins 2 min after activation and exceeds resting levels by 5-10 min. Cofilin is dephosphorylated with identical kinetics but fails to become rephosphorylated when platelets are stimulated under stirring conditions. Cofilin is normally rephosphorylated when platelets are stimulated in the presence of Arg-Gly-Asp-Ser (RGDS) peptide or wortmannin to block alpha(IIb)beta3 cross-linking and signaling or in platelets isolated from a patient with Glanzmann thrombasthenia, which express only 2-3% of normal alpha(IIb)beta3 levels. Furthermore, actin assembly and Arp2/3 complex incorporation in the platelet actin cytoskeleton are decreased when alpha(IIb)beta3 is engaged. Our results suggest that cofilin is essential for actin dynamics mediated by outside-in signals in activated platelets.