Vascular smooth muscle cell (VSMC) differentiation and phenotypic modulation is characterized by changes in mRNA expression for smooth muscle (SM) marker contractile proteins such as alpha-SM actin and SM22 alpha. Transforming growth factor beta1 (TGF-beta 1) is a potent VSMC differentiation factor; however, it is not known if other TGF-beta-superfamily members, in particular the bone morphogenetic proteins (BMPs), modulate VSMC phenotype. Here we demonstrate that a large subset of TGF-beta-superfamily members and their type I receptors are differentially co-expressed as VSMC phenotype changes during fetal/neonatal development and that BMP2, -4, and -6 reciprocally regulate SM-marker mRNA and protein expression in vitro. BMP2 and BMP6 decrease expression of the SM markers alpha-SM actin, SM22alpha, and calponin in rat VSMCs, whereas BMP4 increases their expression. The effects of BMP-2, -4, and -6 on SM marker gene transcription are mediated through a consensus TGF-beta-controlling element, the TCE, which is common to regulatory regions of SM-marker genes. Moreover, co-treatment experiments revealed that BMP-2, -4, and -6 each inhibit TGF-beta 1-modulated increases in SM22alpha reporter gene activity. Regardless of whether they positively or negatively regulate SM marker expression, TGF-beta 1 and BMP-2, -4, and -6 all induced binding of the Krüppel-like transcription factor, GKLF/KLF4, to the TGF-beta control element. Induction of KLF4 was confirmed by immunocytochemistry and Western blotting, which revealed that a lower molecular weight KLF4 protein is induced after treatment with TGF-beta-superfamily members. Taken together, our results demonstrate that multiple members of the TGF-beta superfamily act in concert to modulate VSMC phenotype.