Lipid accumulation in pancreatic beta-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the beta-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.