Virtually all smooth muscle genes analyzed to date contain two or more essential binding sites for serum response factor (SRF) in their control regions. Because SRF is expressed in a wide range of cell types, it alone cannot account for smooth muscle-specific gene expression. We show that myocardin, a cardiac muscle- and smooth muscle-specific transcriptional coactivator of SRF, can activate smooth muscle gene expression in a variety of nonmuscle cell types via its association with SRF. Homodimerization of myocardin is required for maximal transcriptional activity and provides a mechanism for cooperative activation of smooth muscle genes by SRF–myocardin complexes bound to different SRF binding sites. These findings identify myocardin as a master regulator of smooth muscle gene expression and explain how SRF conveys smooth muscle specificity to its target genes.