Mesangial cells express smooth muscle alpha-actin (SM alpha-actin) in response to glomerular injury in vivo, and SM alpha-actin gene expression serves as a genetic marker characterizing the activated phenotype of mesangial cells. We used a molecular genetic approach to analyze the SM alpha-actin promoter and evaluate transcriptional mechanisms that might direct the genetic switch of mesangial cells to the activated phenotype. The sequence spanning -894 to +1 of the SM alpha-actin promoter directed high levels of transcription that were attenuated in serum-restricted cells and upregulated upon treatment with serum or endothelin-1. Deletional analysis revealed a core promoter fragment, from positions -122 to +1, that was necessary and sufficient for transcription. This core activity was modulated by upstream sequences between -670 and -122. The 122-bp core promoter contains two highly conserved CArG box motifs (designated CB1 and CB2), and introduction of deletion mutations of either CB1 or CB2 reduced transcription in mesangial cells to near basal levels. Further analysis revealed that CB1 and CB2 acted synergistically when subcloned upstream of a heterologous, minimal thymidine kinase promoter. CB2 alone was sufficient to confer serum inducibility to a heterologous promoter, but both CB2 and CB1 were required for maximal levels of serum-induced transcription. Collectively, these results demonstrate that CB1 and CB2 cooperate to mediate serum-induced activation of the SM alpha-actin promoter in mesangial cells.