The effects of various nucleosides and nucleotides upon glucagon secretion from the isolated perfused rat pancreas were studied. Increasing glucagon secretion was found with increasing concentrations of exogenous cyclic AMP (2 X 10(-4) M, 2 X 10(-3) M and 1 X 10(-2) M). Stimulation of alpha cell secretion was also found with 2 X 10(-3) M 2'AMP, 3'AMP, 5'AMP, ADP, Adenosine, NADP, and NADPH. One X 10(-3) M cyclic GMP elicited significant glucagon secretion. The pattern of glucagon release was similar in all cases with peak secretion occurring during the 30- to 90-s time period following initiation of the stimulus. No significant increase of glucagon secretion was found in response to ATP, guanosine, 2'GMP, 3'GMP, 5'GMP, GTP, xanthosine, inosine, adenine, xanthine, thymidine, cytidine, ribose, nicotinamide, and uric acid. On the basis of the above results, the structural requirement for stimulation of glucagon secretion appears to be adenine linked to ribose, with phosphate groups being unnecessary. The conclusion of this study is that a new class of compounds capable of stimulating glucagon secretion has been identified, and important questions are thus raised about the mechanism of the action of exogenous cyclic AMP.