Actin filaments carry out diverse cellular functions including cell division, intracellular transport, and muscle contraction. Tropomyosin (Tm) is an α-helical coiled-coil protein that regulates actin in most eukaryotic muscle and nonmuscle cells. Nonmuscle isoforms of Tm, particularly Tm5NM1 (Tpm3.1) (short isoform, 247 a.a., TPM3 gene), play an important role in cellular functions such as cell migration, and cytokinesis as well as in the transformation and metastasis of cancer cells. In previous work, mutation of evolutionarily-conserved residues in striated muscle αTm (Tpm1.1) (long isoform, 284 a.a., TPM1 gene) revealed the regions important for actomyosin regulation. In the present study, we have mutated evolutionarily-conserved residues in Tpm3.1 to determine the molecular basis for isoform-specificity of actomyosin regulation by Tms. We mutated surface residues in nonmuscle Tpm3.1 in periods P1-P7 at positions homologous to the residues that are important for actomyosin regulation by striated muscle Tpm1.1 (Barua et al., 2012). In vitro motility assays were carried out to determine the effect of mutations on actin filament velocities. Actin-Tm velocities with skeletal myosin are inhibited by Tpm1.1 (∼60%) but activated by Tpm3.1 (∼60%) relative to actin alone. The Tpm3.1 mutants had little or no effect on velocity, except for the P3 and P6 mutants that showed a ∼50-60% inhibition in filament velocity relative to WT Tpm3.1. In comparison, amongst the Tpm1.1 mutants, the P3 and P6 mutants also showed the largest inhibition (∼70-80%) in filament velocity relative to WT Tpm1.1. These results indicate that the same regions of Tm (periods 3 and 6) are important for regulation of skeletal myosin by two different isoforms that have contrasting effects (inhibition by Tpm1.1 vs. activation by Tpm3.1) on filament velocity. Supported by NIH.