1. Insulin secretion from pieces of rabbit pancreas incubated in vitro was studied in media of different ionic compositions.2. Insulin secretion stimulated by glucose was abolished by removal of calcium from the incubation medium and inhibited by a twofold rise in the calcium concentration to 10.2 m-equiv/l. Removal of magnesium from the medium did not affect glucose-stimulated secretion but a tenfold rise in the extracellular magnesium concentration to 24 m-equiv/l. abolished secretion.3. The replacement of calcium by an equivalent amount of barium (5.1 m-equiv/l.) stimulated insulin secretion. Barium stimulation declined with time and was inhibited by the presence of calcium, 5.1 m-equiv/l., or abolished by the presence of magnesium, 24 m-equiv/l., in the incubation medium.4. The interactions of mono- and divalent cations on insulin secretion were studied by using ouabain or potassium as tools to raise intracellular sodium concentration and barium as a calcium analogue. Ouabain and potassium were effective stimuli only in the presence of calcium, and barium only stimulated insulin secretion in the presence of sodium.5. The results of these experiments suggest that both calcium and sodium must act at the beta cell membrane or enter the cell before insulin release can occur in response to a variety of stimuli.