Smooth and skeletal muscle tissues are composed of distinct cell types that express related but distinct isoforms of the structural genes used for contraction. These two muscle cell types are also believed to have distinct embryological origins. Nevertheless, the phenomenon of a phenotypic switch from smooth to skeletal muscle has been demonstrated in several in vivo studies. This switch has been minimally analyzed at the cellular level, and the mechanism driving it is unknown. We used immunofluorescence and RT-PCR to demonstrate the expression of the skeletal muscle-specific regulatory genes MyoD and myogenin, and of several skeletal muscle-specific structural genes in cultures of the established rat smooth muscle cell lines PAC1, A10, and A7r5. The skeletal muscle regulatory gene Myf5 was not detected in these three cell lines. We further isolated clonal sublines from PAC1 cultures that homogeneously express smooth muscle characteristics at low density and undergo a coordinated increase in skeletal muscle-specific gene expression at high density. In some of these PAC1 sublines, this process culminates in the high-frequency formation of myotubes. As in the PAC1 parental line, Myf5 was not expressed in the PAC1 sublines. We show that the PAC1 sublines that undergo a more robust transition into the skeletal muscle phenotype also express significantly higher levels of the insulin-like growth factor (IGF1 and IGF2) genes and of FGF receptor 4 (FGFR4) gene. Our results suggest that MyoD expression in itself is not a sufficient condition to promote a coordinated program of skeletal myogenesis in the smooth muscle cells. Insulin administered at a high concentration to PAC1 cell populations with a poor capacity to undergo skeletal muscle differentiation enhances the number of cells displaying the skeletal muscle differentiated phenotype. The findings raise the possibility that the IGF signaling system is involved in the phenotypic switch from smooth to skeletal muscle. The gene expression program described here can now be used to investigate the mechanisms that may underlie the propensity of certain smooth muscle cells to adopt a skeletal muscle identity.