Muscle differentiation is characterized by the induction of genes encoding contractile structural proteins and the repression of nonmuscle isoforms from these gene families. We have examined the importance of this regulated order of gene expression by expressing the two sarcomeric muscle actins characteristic of the differentiated state, i.e. alpha-skeletal and alpha-cardiac actin, in C2 mouse myoblasts. Precocious accumulation of transcripts and proteins for a group of differentiation-specific genes was elicited by alpha-skeletal actin only: four muscle tropomyosins, two muscle actins, desmin and MyoD. The nonmuscle isoforms of tropomyosin and actin characteristic of the undifferentiated state continued to be expressed, and no myosin heavy or light chain or troponin transcripts characteristic of muscle differentiation were induced. Stable transfectants displayed a substantial reduction in cell surface area and in the levels of nonmuscle tropomyosins and beta-actin, consistent with a relationship between the composition of the actin cytoskeleton and cell surface area. The transfectants displayed normal cell cycle progression. We propose that alpha-skeletal actin can activate a regulatory pathway linking a subset of muscle genes that operates independently of normal differentiation and withdrawal from the cell cycle.