Five peptide analogs of the actomyosin ATPase inhibitory region of troponin I (Tn-I) have been synthesized by the solid-phase method. One analog was constructed with a sequence identical to that found in species of Tn-I isolated from chicken fast, rabbit fast and rabbit slow skeletal muscle. A second peptide was made identical to the sequence of the homologous inhibitory region of Tn-I from rabbit cardiac muscle. The remaining three analogs were hybrids of the two sequences. We have verified that rabbit skeletal fast Tn-I was a better inhibitor than rabbit cardiac Tn-I in a rabbit skeletal actomyosin assay system and extended these studies to show that the same results were found in an assay system which used rabbit cardiac actomyosin. Our skeletal fast muscle peptide analog was also a better inhibitor in both assay systems than the cardiac Tn-I peptide analog. The hybrid peptides indicated that the changing of proline 110 to a threonine residue had no effect and suggested that position 110 was not essential to inhibition. The other amino acid change, the replacement of arginine 113 by a leucine residue, was shown to be the crucial difference and resulted in lower activity. Thus, th differences in relative inhibitory activity of rabbit skeletal fast and cardiac Tn-I can be at least partially and possibly solely explained by the single amino acid substitution at position 113. Short active sequences of proteins, like the Tn-I inhibitory region are potentially of enormous value as probes of structure-function relationships.