Vertebrate nonmuscle cells, such as human and rat fibroblasts, express multiple isoforms of tropomyosin, which are generated from four different genes and a combination of alternative promoter activities and alternative splicing. The amino acid variability among these isoforms is primarily restricted to three alternatively spliced exon regions; an amino-terminal region, an internal exon, and a carboxyl-terminal exon. Recent evidence reveals that these variable exon regions encode amino acid sequences that may dictate isoform-specific functions. The differential expression of tropomyosin isoforms found in cell transformation and cell differentiation, as well as the differential localization of tropomyosin isoforms in some types of culture cells and developing neurons suggest a differential isoform function in vivo. Tropomyosin in striated muscle works together with the troponin complex to regulate muscle contraction in a Ca(2+)-dependent fashion. Both in vitro and in vivo evidence suggest that multiple isoforms of tropomyosin in nonmuscle cells may be required for regulating actin filament stability, intracellular granule movement, cell shape determination, and cytokinesis. Tropomyosin-binding proteins such as caldesmon, tropomodulin, and other unidentified proteins may be required for some of these functions. Strong evidence for the distinct functions carried out by different tropomyosin isoforms has been generated from genetic analysis of yeast and Drosophila tropomyosin mutants.