A hallmark of smooth muscle cells (SMCs) in culture and the injured vessel wall is their phenotypic modulation from a differentiated state to one of heightened growth, migration, and matrix synthesis. The transcriptional mechanisms underlying this altered genetic program have yet to be elucidated. Serum response factor (SRF) has emerged as a critical regulator of SMC-restricted gene expression via its interaction with proximal CArG elements; however, levels of SRF protein do not change during SMC phenotypic modulation, suggesting a role for other factors or events in this process. One such factor could be myocardin, a novel SRF coactivator recently cloned from cardiac tissue. Levels of myocardin are abundantly expressed in rat aortic media along with key SMC-restricted genes. In several SMC lines, myocardin mRNA levels decrease in parallel with the loss or attenuation of SMC marker expression. Transient transfection experiments with CMV-driven myocardin in both SMC and non-SMC reveal CArG-dependent transactivation of the SM-Calp promoter-enhancer. Several additional CArG-dependent SMC promoters show variable activation in a cell-and promoter-context dependent manner. To determine whether myocardin could activate an endogenous program of SMC differentiation, we stably transfected L6 myoblasts and assessed SMC marker expression and growth. Results reveal the expression of several SMC markers concomitant with a lower growth potential. Collectively, these studies suggest that myocardin is an important component of a molecular switch for the SMC differentiation program.