The role of cAMP in the inhibitory effect of epinephrine [E] on insulin secretion was studied by comparing the dose-response relationships of the effects of E on insulin release and [3H]cAMP content in rat pancreatic islets prelabeled with [2-3H]adenine and stimulated by various agents in vitro. Glucagon-stimulated insulin release and increased [3H]cAMP in islets were inhibited in a parallel dose-dependent fashion by E. In contrast, glucose-stimulated insulin release was more sensitive to inhibition by E than was glucose-induced [3H]cAMP accumulation. Thus 3..times. 10-8 M E produced 50% inhibition of the insulin release stimulated by 26.7 mM glucose, whereas 10-6 M E was required to produce 50% inhibition of the [3H]cAMP response to glucose. In contrast to the inhibitory effects of a high concentration of E (10-6 M) on glucagon and glucose-stimulated increases in islet [3H]cAMP, this concentration of E did not decrease the increased [3H]cAMP levels in islets incubated with either the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1.0 mM) or the adenylate cyclase stimulator, cholera toxin (10 .mu.g/ml). Phentolamine (10-5 M) abolished the inhibitory effects of 10-6 M E on both the [3H]cAMP and the insulin response to glucose. E may inhibit glucose-induced insulin release by a dual .alpha.-adrenergic action: at low concentrations, E may inhibit insulin release by a mechanism (s) distinct from the adenylate cyclase-cAMP system; and at high concentrations, E may further inhibit insulin release by interfering with the ability of glucose to increase cAMP in the pancreatic .beta. cell rather than by any direct effect on adenylate cyclase.