Ligation of endothelial cell (EC) CD40 induces the expression of several proinflammatory cytokines as well as angiogenesis factors, including vascular endothelial growth factor (VEGF). Moreover, despite the reported importance of CD40 in cell-mediated immunity, little is known of the CD40-induced signaling pathways in EC. In this study, we have investigated the function of the Ras signaling pathway(s) for CD40-induced overexpression of VEGF. EC were transiently transfected with a full-length VEGF promoter-luciferase construct and a dominant-inhibitory mutant of Ras (Ras17N). Following transfection, ligation of CD40 with soluble CD40 ligand resulted in a significant increase in VEGF transcriptional activation, and the inhibitory mutant of Ras blocked this CD40-induced VEGF overexpression. Using EMSA and Western blot analysis, we demonstrated that CD40-dependent binding of nuclear protein(s) to the VEGF promoter and CD40-induced VEGF protein expression in EC were also inhibited by the Ras mutant. Immunoprecipitation studies revealed that ligation of CD40 on EC promoted an increased association of Ras with its effector molecules Raf, Rho, and phosphatidylinositol 3-kinase (PI3K). But, cotransfection of effector-loop mutants of Ras determined that only PI3K was functional for Ras-induced VEGF transcription. Also, wortmanin and a dominant-inhibitory mutant of PI3K inhibited CD40-induced overexpression of VEGF. Together these findings demonstrate that both Ras and PI3K are intermediaries in CD40-induced regulation of VEGF in EC. We believe our findings are of importance in several chronic inflammatory diseases, including atherosclerosis and allograft rejection associated with both CD40-CD40 ligand signaling as well as VEGF expression and function.