Cyclic AMP potentiates glucose-stimulated insulin release by actions predominantly at a site, or sites, distal to the elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). Glucose also acts at a site, or sites, distal to the elevation of [Ca2+]i via the ATP-sensitive K+ channel (K+ATP channel)-independent signaling pathway. Accordingly, using rat pancreatic islets, we studied the location of the action of cAMP and its interaction with the glucose pathway. Forskolin, an activator of adenylyl cyclase, raised intracellular cAMP levels and enhanced KCl-induced (Ca2+ -stimulated) insulin release in the presence, but not in the absence, of glucose. Thus, cAMP has no direct effect on Ca2+ -stimulated insulin release. The interaction between cAMP and glucose occurs at a step distal to the elevation of [Ca2+]i because forskolin enhancement of KCl-induced insulin release, in the presence of glucose, was demonstrated in the islets treated with diazoxide, a K+ATP channel opener. The enhancement of insulin release was not associated with any increase in [Ca2+]i. Furthermore, the interaction between cAMP and glucose was unequivocally observed even under stringent Ca2+ -free conditions, indicating the Ca2+ -independent action of cAMP. This action of cAMP is physiologically relevant, because not only forskolin but also glucagon-like peptide 1, glucose-dependent insulinotropic polypeptide, and pituitary adenylyl cyclase activating polypeptide exerted similar actions. In conclusion, the cAMP/protein kinase A pathway has no direct effect on Ca2+ -stimulated insulin exocytosis. Rather, it strongly potentiates insulin release by increasing the effectiveness of the K+ATP channel-independent action of glucose.