E2F is a family of transcriptional factors that control G₁/S transition. We investigated how the E2F family participates in the biological responses of vascular smooth muscle cells (VSMC) to vasoconstrictive hormones compared with fetal bovine serum (FBS). FBS induced upregulation of E2F-1 and E2F-5 at both mRNA and protein levels and slightly reduced E2F-3 protein. Angiotensin II (ANG II) and arginine vasopressin increased E2F-3 protein, but not E2F-1 and E2F-5, without upregulating its mRNA level. FBS transactivated the E2F-1 gene through the induction of free E2F-1 binding onto its promoter, whereas ANG II-induced binding of E2F-3 did not result in activation of the E2F-1 promoter. These changes are responsible for hypertrophic or hyperplastic response of VSMC to different growth factors or stimulants. In contrast, both FBS and vasoconstrictive hormones drove transcription of the cdc6 gene by downregulating p130 and recruiting free E2F-3 in the latter, which underlies the progression of VSMC into S phase.