The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the β-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human β-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the β-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of β-actin gene expression was mediated at least in part through one of the three β-actin CCArGG elements. © 1995 by The Endocrine Society.