Actin filaments are required for diverse cellular functions including cell division, intracellular transport, and muscle contraction. Tropomyosin (Tm) is a α-helical coiled-coil protein that regulates its functions and stability in muscle and nonmuscle eukaryotic cells. Nonmuscle isoforms of Tm and particularly, Tm5NM1 (a γTm, product of TPM3), have been found to play a role in the transformation and metastasis of cancer cells in addition to being important for normal cellular functions such as cell migration, cell division and organelle transport. In previous work, by making mutations at evolutionarily conserved residues in a striated muscle αTm, residues important for actin binding and myosin regulation were identified. In the present study, we have mutated conserved residues in Tm5NM1 to determine the molecular basis of isoform-specificity for functions such as actin binding and actomyosin regulation. We mutated surface residues in muscle αTm and Tm5NM1 that differed between the two isoforms at homologous positions in periods P1 and P7 of αTm to determine the effect of sequence differences on function. We also mutated residues in Tm5NM1 in periods P5 and P6 at homologous positions to those that were shown to be important for myosin regulation in muscle αTm in a previous study. In vitro motility assays for determining myosin regulation were carried out. Actin-Tm filament velocities were inhibited by αTm (∼60%) but activated by Tm5NM1 (∼50%) relative to actin alone. The αTm P1 and P7 mutants showed ∼40-70% increase in velocity compared to WT αTm. The Tm5NM1 mutants had no effect on velocity relative to WT Tm5NM1, except the P6 mutant that showed a ∼50% decrease in velocity. Therefore, αTm and Tm5NM1 have differential effects on actomyosin regulation depending on the site of mutation. Supported by NIH and Aresty Research Center at Rutgers.