Protein kinase A (PKA) -independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K+ channels (K-ATP) was provided by one prior study of human beta cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analog (ESCA) inhibited a sulfonylurea-sensitive K+ current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human beta cells and rat INS-1 cells, we now report that 2'-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K-ATP channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2'-O-Me-cAMP (50 micromolar), the dose-response relationship describing ATP-dependent inhibition of K-ATP channel activity is left-shifted such that the concentration of ATP producing 50% inhibition (IC50) is reduced from 22 micromolar to 1 micromolar for human beta cells, and from 14 micromolar to 4 micromolar for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 micromolar), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC50 12 micromolar for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2'-O-Me-cAMPS) also inhibits K-ATP channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human beta cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP-sensitivity of K-ATP channels is regulated by Epac.