Actin is of fundamental importance to all eukaryotic cells. Of the six mammalian actins, beta (beta) and gamma (gamma) cytoplasmic are the isoforms found in all nonmuscle cells and differ by only four amino acids at the amino-terminal region. Both genes are regulated temporally and spatially, though no differences in protein function have been described. Using fluorescent double in situ hybridization we describe the simultaneous intracellular localization of both beta and gamma actin mRNA. This study shows that myoblasts differentially segregate the beta and gamma actin mRNAs. The distribution of gamma actin mRNA, only to perinuclear and nearby cytoplasm, suggests a distribution based on diffusion or restriction to nearby cytoplasm. The distribution of beta actin mRNA, perinuclear and at the cell periphery, implicates a peripheral localizing signal which is unique to the beta isoform. The peripheral beta actin mRNA corresponded to cellular morphologies, extending processes, and ruffling edges that reflect cell movement. Total actin and gamma actin protein steady-state distributions were identified by specific antibodies. gamma actin protein was found in both stress fibers and at the cell plasma membrane and does not correspond to its mRNA distribution. We suggest that localized protein synthesis rather than steady-state distribution functionally differentiates between the actin isoforms.