We have quantitatively characterized the interaction of several concentrations of norepinephrine and acetylcholine applied simultaneously on insulin secretion in response to an intermediate glucose concentration (10 mM) from statically incubated pancreatic islets isolated from normal rats. When either norepinephrine or acetylcholine were applied alone, insulin secretion was respectively inhibited or enhanced in a dose-dependent manner. When combinations of norepinephrine and acetylcholine were applied simultaneously to the islet, insulin secretion was enhanced, inhibited, or not affected. The net effects of the interaction of norepinephrine and acetylcholine on insulin secretion were not only a function of the concentration of each neurotransmitter but also the relative concentrations of both neurotransmitters. Analysis of these interaction data suggests that 1) insulin secretion is the result of a complex interaction between sympathetic and parasympathetic neural and metabolic inputs; 2) norepinephrine and acetylcholine modulate insulin secretion primarily by direct action on the beta-cell membrane; 3) insulin secretion is more sensitive to perturbations in norepinephrine concentration than acetylcholine concentrations under these experimental conditions; and 4) the autonomic nervous system has the potential to play a major role in the control of insulin secretion in response to glucose.