How does cyclic AMP potentiate insulin secretion from pancreatic islet beta-cells? This question is fundamental to understanding how hormones such as glucagon, which elevates cAMP, stimulate insulin secretion and so contribute to the normal secretory response of the islet. It is well established that a rise in the cytoplasmic Ca2+ concentration ([Ca2+]i) is essential for insulin secretion and therefore cAMP has been proposed to act by elevating [Ca2+]i. But studies on permeabilized beta-cells indicate that cAMP increases insulin release even when [Ca2+]i is held constant. We have used microfluorimetry and the patch-clamp technique to measure changes simultaneously in Ca2+ currents, [Ca2+]i and exocytosis in a single beta-cell in response to cAMP. We show here that cAMP, through activation of protein kinase A, increases Ca(2+)-influx through voltage-dependent L-type Ca2+ channels, thereby elevating [Ca2+]i and accelerating exocytosis. More importantly, cAMP also promotes insulin release by a direct interaction with the secretory machinery, which accounts for as much as 80% of its effect.