Although it has been established that factor V (FV) becomes associated irreversibly with the platelet cytoskeleton after stimulation with thrombin, the chemical nature of this complex is not known. Factor V has recently been demonstrated to be a substrate for factor XIIIa and to form factor V oligomers. We now show that thrombin-activated 125I-FV specifically links to a single protein (43 kDa) of the solubilized platelet membrane in a reaction which requires Ca++ and factor XIIIa. In a purified system, FV, activated by thrombin, forms covalently linked high molecular complexes with 125I-actin catalyzed by factor XIIIa. The site of crosslinking of actin was the factor V fragments, 150 kDa (connecting peptide, C1) and its parent molecule 200 kDa (B). Using radiolabeled actin and unlabeled FV, factor XIIIa catalysed the formation of both homopolymers and heteropolymers. Unlabeled actin was found to compete with radiolabeled FV as a substrate for FXIIIa. To evaluate the biological significance of the crosslinking of factor V to actin, intact platelets were treated with B10 (monoclonal antibody to C1), or monospecific polyclonal antibodies to actin or FXIII. After stimulation with thrombin, the cytoskeleton (material insoluble in Triton X-100) showed markedly decreased 125I-FV in the crosslinked complexes. FV coagulant activity associated with platelet cytoskeleton was also diminished following incubation with an antibody to actin, factor XIII, or B10. These data suggest that FV, through the C1 domain, is crosslinked to actin in the cytoskeleton of thrombin-treated platelets. Activated factor XIII may play a role in plasma FV-platelet interaction as well as the expression of FV derived from the alpha-granules on the cytoskeleton during platelet stimulation.