Expression of -smooth muscle actin (-SMA) defines the phenotype of activated (myofibroblastic) hepatic stellate cells. These cells, but not quiescent stellate cells, have a high level of -SMA and c-Myb expression, as well as increased c-Myb-binding activities to the proximal -SMA E box. Therefore, we analyzed the role of c-Myb in -SMA transcription and stellate cell activation. Activated primary rat stellate cells displayed a high expression of the 724 and 271 -SMA/luciferase (LUC) chimeric genes, which contain c-Myb binding sites (223/216 bp). -SMA/LUC minigenes with mutation (219/217 bp), truncation (224 bp), or deletion (191 bp) of the c-Myb binding site were not efficiently transcribed. Transfection of wild-type c-Myb into quiescent stellate cells, which do not express endogenous c-Myb, induced a ~10-fold stimulation of 724 -SMA/LUC expression. Conversely, expression of either a dominant-negative c-Myb basic domain mutant (Cys⁴³  Asp) or a c-Myb antisense RNA blocked transcription from the 724 -SMA/LUC or 271 -SMA/LUC in activated cells. Moreover, transfection of c-myb antisense, but not sense, RNA inhibited both expression of the endogenous -SMA gene and stellate cell activation, whereas transfection of c-myb stimulated -SMA expression in quiescent stellate cells. These findings suggest that c-Myb modulates the activation of stellate cells and that integrity of the redox sensor Cys⁴³ in c-Myb is required for this effect.