The effect of calcium and magnesium ions on glucagon and insulin secretion was studied in vitro, using pieces of pancreas from duct-ligaturated rats. Omission of calcium from the incubation media stimulated glucagon release. Addition of barium to the calcium-depleted media did not prevent the stimulation of glucagon release. Omission of magnesium also tended to increase the rate of glucagon secretion. Much higher rates of glucagon release were observed in the concomitant absence of calcium and magnesium, or in calcium-depleted media supplemented with a chelating agent (EGTA). On the other hand, excess of calcium (8 mEq/1) was without effect, and excess magnesium (20 mEq/1) inhibited basal or arginine-induced glucagon release. In these various experimental conditions, except those in which a marked stimulation of glucagon occurred (absence of both cations, presence of EGTA), the rate of insulin secretion followed the patterns documented by previous investigators. It is concluded that calcium and magnesium, in contrast to their effect on the β cell, act synergistically upon the secretory process of the α2 cell in such a way that glucagon release is stimulated at low levels of both cations. (Endocrinology93: 1360, 1973)