We have designed a series of recombinant peptides derived from the C-terminus of human caldesmon (amino acids 663-793, domain 4) to determine the structural basis of the multiple-sited caldesmon-actin-tropomyosin interaction. All the recombinant peptides are able to bind to actin and inhibit actin-activated myosin ATPase activity; 1 mol of peptide is bound per actin for >90% inhibition. However, equivalent inhibition of actin-tropomyosin activation of myosin ATPase requires less than one peptide per seven actin to be bound. We have found two sequences, H2 (amino acids 683-767) and H2+12 (amino acids 683-779), from the center of domain 4 which potentiate actin-tropomyosin filament activity; i.e., their effect is opposite to caldesmon. Maximum potentiation correlates with one H2 or H2+12 bound per four actin. This effect is completely dependent upon the presence of tropomyosin on the actin filament. H2 and H2+12 also increase actin-tropomyosin filament velocity in the in vitro motility assay. If the H2 sequence is extended by 20 amino acids at the N-terminal end to the N-terminus of domain 4, the peptide becomes an inhibitor. If H2 is extended by 19 amino acids at its C terminus, it becomes a tropomyosin-dependent inhibitor, and with a further extension of 7 amino acids to reach the C-terminus of human caldesmon (H2+26), inhibition is more potent. We conclude that three regions in domain 4 of caldesmon contribute to tropomyosin-dependent inhibition of actomyosin ATPase: a central segment [747-767 (690-710 in the chicken sequence)], which is essential but not sufficient for tropomyosin-dependent inhibition of actomyosin ATPase; and two actin binding segments N-terminal and C-terminal to this segment, 663-682 (606-625) and 770-793 (713-737). If only the central segment is present (H2, H2+12), the actin-tropomyosin-caldesmon peptide complex is not inhibitory, and its properties resemble actin-tropomyosin-caldesmon-Ca2+ x calmodulin.