Atherosclerosis is characterized by hyperplastic neointima and an inflammatory response with cytokines such as TNFa. TNFa is a pleiotropic cytokine that mediates inflammatory, proliferative, cytostatic and cytotoxic effects in a variety of cell types, including endothelial cells and vascular smooth muscle cells (VSMCs). Interestingly, TNFa has been shown to play two very opposing roles in these cell types; it inhibits proliferation and induces apoptosis in endothelial cells, while it enhances the proliferation and migration of VSMCs. Here we show that TNFa is capable of stimulating proliferation of rat VSMCs as well as human VSMCs in a Raf-1/MAP K-dependent manner. TNFa could increase the expression of E2F-regulated proliferative cdc6, Thymidylate synthase (TS) and cdc25A genes in Aortic smooth muscle cells (AoSMC), as seen by real time PCR assays. There is an activation of the stress-induced kinase, JNK1, in VSMCs upon TNFa stimulation. TNFa was capable of inducing binding of the Raf-1 kinase to Rb, and treatment with the Rb-Raf-1 inhibitor, RRD-251, could prevent TNFa-induced S-phase entry in AoSMCs. In addition, inhibition of Raf-1 or Src kinases using pharmacologic inhibitors could also prevent S-phase entry, while inhibition of JNK was not as effective. These results suggest that inhibiting the Rb-Raf-1 interaction is a potential avenue to prevent VSMC proliferation associated with atherosclerosis.