We report here that endogenous prostaglandin E2 (PGE2) resulting from cyclooxygenase (COX)-2 expression in a highly metastatic murine breast cancer cell line C3L5 upregulates IFN-γ + LPS-induced nitric oxide (NO) synthase (iNOS) expression and NO production. This action of PGE2 is mediated through the EP4 receptor in a cAMP-dependent manner. Both nonselective and selective COX-2 inhibitors suppressed IFN-γ + LPS-induced NO production, which was largely restored by exogenous PGE2 or EP4 receptor agonist PGE1 alcohol. EP4 antagonist AH-23848B inhibited NO production with a concomitant downregulation of iNOS mRNA in IFN-γ + LPS-stimulated cells. cAMP dependence of NO production by cells under inducible conditions was demonstrated by the use of known modulators of intracellular cAMP. Since both COX-2 and iNOS are implicated in breast cancer progression, our findings of EP4 receptor-mediated upregulation of iNOS in COX-2-expressing breast cancer cells suggest that blocking COX-2 and/or EP4 may provide a simple therapeutic modality in this tumor model. © 2003 Wiley-Liss, Inc.