Abstract Exchange protein directly activated by cAMP (Epac1 and Epac2) are recently identified targets for cAMP signaling. Epac activates Rap1 by catalyzing the conversion of GDP-Rap1 to GTP-Rap1, which is independent of classical cAMP/PKA signaling. In this study, we examined the activation of Epac/Rap1 signaling by prostaglandin E2 (PGE2), a proinflammatory lipid mediator, in synovial fibroblasts (RASF) from rheumatoid arthritis (RA) patients. Expression of Epac1 mRNA and protein were observed in RASF, whereas Epac2 expression was absent. Rap1 was also expressed and its active form, GTP-Rap1, was rapidly increased by PGE2 and it was further enhanced in the presence of IBMX, a phosphodiesterase inhibitor. In addition, the effect of PGE2 on Rap1 was mimicked by forskolin, (a direct activator of adenylate cyclase) and butaprost (an EP2 agonist) but not by sulprostone (an EP1 & EP3 agonist), which were correlated to elevation of cAMP. Furthermore, Rap1 activation was also observed by 8-CPT-2Me-cAMP (an Epac agonist) as well as 6-Bnz-cAMP (a PKA agonist). Epac1/Rap1 signaling via EP receptor associated to the elevation of cAMP may act in concert with classical PKA signaling after exposure to PGE2. Epac1/Rap1 signaling may be an alternate signaling pathway in addition to PKA signaling for PGE2 mediated pathophysiology of chronic inflammatory diseases such as RA. Grant Support: Arthritis Foundation Biomedical Sciences Grant and NIH/NIAMS AR49010.