Talin, a putative homodimer of 230-kDa polypeptides, was cleaved into the N-terminal 47-kDa and C-terminal 190-kDa fragments with calpain II. The 190-kDa fragment, but not the 47-kDa fragment, was found to bind to actin. The 190-kDa fragment possessed similar levels of activities to stimulate both polymerization of G-actin and alpha-actinin-dependent gelation of F-actin as did intact talin. Limited digestions of the 190-kDa fragment with chymotrypsin and papain resulted in partial and complete reductions, respectively, of both activities, although these digests contained 95- and 46-kDa major polypeptides, respectively, which were able to bind to actin. Whereas the 190-kDa fragment generated fully cross-linked oligomeric polypeptides on treatment with 1-ethyl-3[3-(dimethylamino)-propyl]carbodiimide, the 95-kDa chymotryptic polypeptide generated heterologous polypeptides cross-linked partially with smaller polypeptides. The papain digest did not contain any cross-linkable polypeptide. Intact talin and the 47-kDa calpain fragment, but not the 190-kDa calpain fragment, were found to bind to phospholipid vesicles containing phosphatidylserine. These results indicate that the N-terminal and C-terminal domains play distinct roles in interacting with the membrane and cytoskeletal elements, respectively, and that the dimeric structure is also required for the latter interactions.