The effects of 5 mM theophylline on L-phenylalanine, L-alanine, and L-leucine-induced insulin secretion were studied using the isolated perfused rat pancreas, varying the sequence of the stimuli. Theophylline in the absence or presence of 3 mM D-glucose caused a small amount of insulin release with a slow onset and a slight and steady increase. Theophylline had no effect on phenylalanine (5, 10, or 20 mM)-induced insulin release independent of the sequence of stimuli. Alanine (20 mM), in the absence of glucose, had no significant insulin stimulatory action. When theophylline was added during the alanine perfusion only a small insulin release, comparable to that given by theophylline alone, could be observed. However, superimposing alanine on a theophylline perfusion led to a potentiation of insulin release. The leucine-induced insulin secretion was significantly altered by the addition of theophylline. At a low concentration of leucine (5 mM) theophylline caused potentiation of leucine-induced insulin secretion. At 10 and 20 mM leucine, theophylline led to a rapid concentration-dependent inhibitory period, followed by a potentiation in the case of 10 mM leucine, and by a restoration of the secretion rate at 20 mM leucine which did not exceed the secretion rate of 20 mM leucine alone. Subsequent removal of theophylline caused a marked "off effect." When 5 or 20 mM leucine was superimposed on a theophylline perfusion, a marked dose-dependent potentiation of the biphasic leucine-induced insulin release and no inhibitory phase could be observed. From these data it must be concluded that the effect of theophylline on the insulin secretory reposure of the beta cell to theophylline and the stimulus. Possible explanations for these phenomena are discussed.