Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.