The relationship between glucagon and insulin secretion was studied using the isolated perfused rat pancreas. Glucagon was found to have no stimulatory effect on the beta cells when glucose was absent from the perfusing medium. There invariably occurred, however, a substantial increase in the amount of insulin secreted (over and above that which was released in response to glucose alone) whenever glucagon and glucose were simultaneously administered to a pancreatic preparation. This potentiation of insulin release also occurred when tolbutamide was substituted, for glucose as the stimulatory agent, thus indicating that glucagon may be active on some end step in the secretory process rather than being mediated via a metabolite of glucose. The action of glucagon was also found to occur regardless of whether or not puromycin was present in the perfusate (at a concentration which has previously been shown to inhibit insulin synthesis), suggesting that a major action of glucagon is to augment the release of preformed insulin.