Myofibroblasts are granulation tissue fibroblasts bearing ultrastructural and biochemical features of smooth muscle cells, such as cytoplasmic microfilaments and alpha-smooth muscle actin expression. They appear transiently during wound healing and more permanently during several pathologic situations such as fibrotic diseases. Transforming growth factor-beta1 has been suggested to be an important promoter of the myofibroblastic phenotype. Here we show that (1) transforming growth factor-beta2, like transforming growth factor-beta1, induces myofibroblast formation in vivo and in vitro; (2) transforming growth factor-beta3 acts as a negative regulator of the myofibroblastic phenotype in vivo but not in vitro; and (3) in vitro, the three different transforming growth factor-beta isoforms are equally able to induce alpha-smooth muscle actin messenger RNA and protein expression in growing and quiescent cultured human and rat subcutaneous tissue fibroblasts. These data confirm that in vitro the behavior of the three different transforming growth factor-beta isoforms is similar, whereas in vivo transforming growth factor-beta isoforms possibly play different but complementary roles in myofibroblast modulation during wound repair.