—Transforming growth factor-β (TGF-β) has been shown to stimulate smooth muscle (SM) α-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-β control element (TCE) located within the first 125 bp of the SM α-actin promoter were required for TGF-β inducibility of SM α-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-β induction of SM α-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-β can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22α, h₁ calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-β inducibility of SM α-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from −155 completely repressed TGF-β responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-β enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-β also stimulated expression of the SMC differentiation marker SM22α in non-SMCs. However, in contrast to SMCs, TGF-β did not induce expression of h₁ calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-β–induced expression of SM α-actin in SMCs and non-SMCs that is modified by a complex interplay of positive- and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-β stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-β alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.