The actin-myosin head complex in the rigor state reveals several high-affinity sites on the actin molecule in sequences 18-28 and 40-113. In the presence of Mg(2+)-ATP, participation of the actin N-terminal 1-7 sequence is known to occur. The proximity of the C-terminal region of actin to the A1 light chain of the myosin head [S-1(A1)] (where S-1 is myosin subfragment-1) was described previously. We observed that C-terminal antigenic structures located near Met-305, Met-325 and Met-355 and the C-terminal end (Cys-374) of actin are markedly modified in the presence of S-1(A1), S-1(A2) and scallop S-1 and in the absence of Mg(2+)-ATP. This seems to rule out any important specific involvement of the A1 light chain in the described conformational changes. An S-1-binding site was located in this actin C-terminal region by testing the tryptic CB9 peptide (360-372 sequence) previously implicated in the A1 light chain interaction. This peptide was able to bind well to S-1(A1), S-1(A2) and scallop S-1, but not in the presence of Mg(2+)-pyrophosphate. These results strengthen the hypothesis of a multisite interface between S-1 and actin located in the actin subdomain I.