E-series prostanoid-4 (EP4) receptor is up-regulated in numerous cancers, including cervical carcinomas, and has been implicated in mediating the effects of PGE₂ in tumorigenesis. In addition to regulation by endogenously biosynthesised PGE₂, neoplastic cervical epithelial cells in sexually active women may also be regulated by prostaglandins present in seminal plasma. In this study we investigated the signal transduction pathways mediating the role of seminal plasma and PGE₂ in the regulation of tumorigenic and angiogenic genes via the EP4 receptor in cervical adenocarcinoma (HeLa) cells. HeLa cells were stably transfected with EP4 receptor in the sense orientation. Seminal plasma and PGE₂ signaling via the EP4 receptor induced the activation of cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) promoters, expression of COX-2 and VEGF mRNA and protein and secretion of VEGF protein into the culture medium. Treatment of HeLa cells with seminal plasma or PGE₂ also rapidly induced the phosphorylation of ERK1/2 via the EP4 receptor. Pre-incubation of cells with a specific EP4 receptor antagonist (ONO-AE2-227) or chemical inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase or mitogen-activated protein kinase kinase (MEK) or co-transfection of cells with dominant negative mutant cDNA targeted against the EGFR, serine/threonine kinase Raf or MEK abolished the EP4-induced activation of COX-2, VEGF and ERK1/2. Therefore we have demonstrated that seminal plasma and PGE₂ can promote the expression of tumorigenic and angiogenic factors, in cervical adenocarcinoma cells via the EP4 receptor, EGFR and ERK1/2 signaling pathways.