1. 1. The effect of noncarbohydrate agents and of alterations in the cationic environment on insulin release has been studied in cultured fetal pancreatic explants of the rat. 2. 2. Tolbutamide alone (81 μg/ml) induced some insulin release. It was clearly more effective, however, in the presence of 11 mM glucose and even more so in the presence of 10 mM caffeine. The smallest effective tolbutamide concentration in the presence of glucose was 9 μg/ml. 3. 3. Glucagon (3.2 μg/ml) elicited a modest insulin release, which was enhanced by addition of 10 mM caffeine and more markedly by 11 mM glucose. The smalelst effective dose in the presence of glucose was 50 ng/ml. A preparation of “gut extract” stimulated the release of insulin in the presence of glucose, while corticotropin and secretin were ineffective. 4. 4. Organic acids were tested at the concentration of 11 mM in the presence of caffeine. Citrate and octanoate enhanced insuling release, whereas glutamate, succinate and fumarate were inhibitory. Neither acetate nor β-hydroxybutyrate altered insulin output. 5. 5. All of the amino acids studied (concentration of 11 mM) stimulated the output of insulin in the presence of caffeine. Lysine was most effective, followed by threonine, arginine and leucine. The least active amino acids were histidine and phenylalanine. 6. 6. The omission or the presence of an excess of K + in the incubation medium stimulated insulin output and, furthermore, enhanced the stimulatory effect of glucose and/or caffeine. Ouabain enhanced the glucose- and caffeine-induced insulin release but only at the relatively high concentration of 1 mM. Lowering of the Na + concentration in the medium suppressed the stimulatory effect of the ommission of K + but was without inhibitory effect at higher K + concentrations. 7. 7. Omitting Ca 2+ from the medium diminished the insulin release induced by glucose and caffeine, either in the absence of K + or in its presence at normal or high concentrations, whereas omitting Mg 2+ had no inhibitory effect. 8. 8. These results were interpreted as consistent with the hypothesis that insulin release from our preparation is dependent, at least in part, upon activation of the adnyl cyclase syste.