To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.