—The aims of the present studies were to define sufficient promoter sequences required to drive endogenous expression of smooth muscle (SM) α-actin and to determine whether regulation of SM α-actin expression in vivo is dependent on CArG (CC(A/T)₆GG) cis elements. Promoter deletions and site directed mutagenesis techniques were used to study gene regulation in transgenic mice as well as in smooth muscle cell (SMC) cultures. Results demonstrated that a Lac Z transgene that contained 547 bp of the 5′ rat SM α-actin promoter was sufficient to drive embryonic expression of SM α-actin in the heart and in skeletal muscle but not in SMCs. Transient transfections into SMC cultures demonstrated that the conserved CArG element in the first intron had significant positive activity, and gel shift analyses demonstrated that the intronic CArG bound serum response factor. A transgene construct from −2600 through the first intron (p2600Int/Lac Z) was expressed in embryos and adults in a pattern that closely mimicked endogenous SM α-actin expression. Expression in adult mice was completely restricted to SMCs and was detected in esophagus, stomach, intestine, lung, and nearly all blood vessels, including coronary, mesenteric, and renal vascular beds. Mutation of CArG B completely inhibited expression in all cell types, whereas mutation of the intronic CArG selectively abolished expression in SMCs, which suggests that it may act as an SMC-specific enhancer-like element. Taken together, these results provide the first in vivo evidence for the importance of multiple CArG cis elements in the regulation of SM α-actin expression.