The interrelationship between Ca2+ and glucose on glucagon release was studied in the in vitro perfused rat pancreas. Spontaneous release during perfusion with glucose-free, Ca-depleted (0.2 meq/l Ca) medium was completely abolished by ethylene glycol bis tetraacetic acid (EGTA; 0.2 mM). Glucose added to Ca-depleted perfusate caused only partial inhibition of glucagon release, even at concentrations of 500 mg/dl, and there was no evidence of a paradoxical increase in secretion with time. When Ca was added in a series of steps (in the absence of additional secretagogues) more than half of the increased glucagon released was elicited by the 1st step (0.5 meq/l). Release patterns at subsequent steps suggested that higher concentrations of Ca may cause mixed stimulation and inhibition. With 70 mg/dl glucose, Ca stimulated release was partially suppressed at all Ca concentrations up through 9 meq/l. With 150 mg/dl glucose, addition of the normally stimulating 0.5 meq/l Ca caused abrupt and complete inhibition of glucagon secretion, and this persisted at all higher Ca concentrations. Insulin release, when high enough to be detected, did not correlate with the glucose/Ca suppression of glucagon. In other experiments, control results and all insulin secretion patterns were qualitatively similar to those reported by other investigators; however, various attempts to demonstrate a paradoxical, increased glucagon secretion by glucose during Ca deprivation were unsuccessful. Small amounts of Ca are normally required for glucagon secretion, although at higher concentrations the effects become complex. Glucagon suppression by glucose is Ca-requiring. Changes in glucagon secretion caused by addition or depletion of Ca can depend on the relative amount of glucose in the milieu.