Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I(sc)) measurement in response to agonist-stimulated Cl- secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 microM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 microM). Both FSK and the Epac activator 8-pCPT-2'-O-Me-cAMP (50 microM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2'-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I(sc) response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2'-O-Me-cAMP on Cl- secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2'-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.