Myofibroblasts are specialized contractile fibroblasts that are critical in wound closure and tissue contracture. Generation of contractile force is correlated with the expression of α-smooth muscle actin (α-SMA); however, little is known regarding molecular mechanisms that control activation of α-SMA in myofibroblasts in granulation tissue. The aims of the present studies were to identify sufficient promoter regions required for α-SMA expression in myofibroblasts in vivo and to determine whether activation of α-SMA expression in myofibroblasts in vivo is dependent on an intronic CArG [CC(A/T)₆GG] and a transforming growth factor-β1 control element (TCE) that are required for α-SMA expression in smooth muscle cells. A Lac Z transgene construct from −2600 through the first intron was expressed in myofibroblasts within granulation tissue of cutaneous wounds in a pattern that closely mimicked endogenous α-SMA expression. Mutation of either the intronic CArG element or the TCE completely inhibited transgene expression in myofibroblasts in granulation tissue and responsiveness to transforming growth factor-β1 in cultured transgenic fibroblasts. These same elements were also critical in regulating α-SMA expression during skeletal muscle repair but not during skeletal muscle development. Taken together, these results provide the first in vivo evidence for the importance of the intronic CArG and TCE cis-elements in the regulation of α-SMA expression in myofibroblasts in granulation tissue.