The role of nutrients and hormones in the regulation of glucagon release is investigated in pancreatic A cells purified by autofluorescence-activated cell sorting. Purified A cells lack secretory activity in 1-h incubation at 1.4 mM glucose. Their release mechanism can be activated by arginine, alanine, and glutamine, alone or in combination. Glucose inhibits amino acid-induced glucagon release through a direct insulin-independent action upon pancreatic A cells. Nutrient-induced glucagon release is suppressed by somatostatin and amplified by (Bu)2cAMP or epinephrine. The epinephrine stimulus is inhibited by 10(-11) M somatostatin and abolished by 10(-10) M of this peptide. The effects of somatostatin and epinephrine are associated with parallel changes in cellular cAMP levels, which is not the case for the variations induced by amino acids or glucose. It is confirmed that calcium is an essential requirement for glucagon release. In contrast to its exquisite sensitivity for somatostatin, the glucagon release process is relatively insensitive to insulin during a 1-h exposure. The hormone affects solely epinephrine-induced glucagon release and its inhibitory action is partial and only observed at 10(-7) M. This suppressive effect of insulin is not attributable to variations in glucose handling but appears associated with the stimulatory effect of epinephrine. It is concluded that a nutrient-induced signal interacts with a hormone-inducible cAMP signal to activate the secretory process in pancreatic A cells.