Vascular smooth muscle cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple smooth muscle-specific genes. In cultured VSMC, vasopressin (AVP) increases, and PDGF decreases expression of smooth muscle -actin (SMA), the earliest marker of SMC. However, it is unknown whether these agents regulate other smooth muscle genes in a similar fashion. SM22 appears secondary to SMA during development, and is also a marker for SMC. This study examined the regulation of SM22 expression by AVP and PDGF in cultured VSMC. Levels of SM22 mRNA and protein were increased by AVP, and suppressed by PDGF. Consistent with these changes, AVP increased SM22 promoter activity, while PDGF inhibited basal promoter activity and blocked the AVP-induced increase. Activation of both JNK and p38 MAP kinase pathways was necessary for AVP-mediated induction of SM22 promoter. Expression of constitutively active Ras produced similar suppressions on SM22 promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS and PI3K activation. Truncational analysis showed that the proximal of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Over-expression of serum response factor enhanced basal and AVP-stimulated promoter activity, but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways, which control expression of multiple smooth muscle genes leading to phenotypic modulation.