SM22α is an adult smooth muscle-specific protein that is expressed in the smooth, cardiac, and skeletal muscle lineages during early embryogenesis before becoming restricted specifically to all vascular and visceral smooth muscle cells (SMC) in late fetal development and adulthood. We have used theSM22αgene as a marker to define the regulatory mechanisms that control muscle-specific gene expression in SMCs. Previously, we reported that the 445-base-pair promoter ofSM22αwas sufficient to direct transcription of a lacZ reporter gene in early cardiac and skeletal muscle cell lineages and in a subset of arterial SMCs, but not in venous nor visceral SMCs in transgenic mice. Here we describe two evolutionarily conserved CArG (CC(A/T)₆GG) boxes in theSM22αpromoter, both of which are essential for full promoter activity in cultured SMCs. In contrast, only the promoter-proximal CArG box is essential for specific expression in developing smooth, skeletal, and cardiac muscle lineages in transgenic mice. Both CArG boxes bind serum response factor (SRF), but SRF binding is not sufficient forSM22αpromoter activity, since overexpression of SRF in the embryonal teratocarcinoma cell line F9, which normally expresses low levels of SRF, fails to activate the promoter. However, a chimeric protein in which SRF was fused to the transcription activation domain of the viral coactivator VP16 is able to activate theSM22αpromoter in F9 cells. These results demonstrate the SM22α promoter-proximal CArG box is a target for the regulatory programs that confer smooth, skeletal, and cardiac muscle specificity to theSM22αpromoter and they suggest that SRF activatesSM22αtranscription in conjunction with additional regulatory factors that are cell type-restricted.