The cytoplasmic Ca2+ concentration ([Ca2+]i) was monitored in individual guinea-pig pancreatic alpha 2-cells exposed to modulators of glucagon release. Addition of the stimulatory amino acid arginine resulted in a sustained increase in [Ca2+]i, whereas the inhibitor glucose had the opposite effect. Epinephrine, the beta-adrenergic agonist isoproterenol, the adenylate cyclase activator forskolin and 8-bromo-cAMP transiently raised [Ca2+]i provided that the cells had been pretreated with glucose. However, simultaneous presence of glucose was not required and the effect occurred even in the absence of extracellular Ca2+. Carbachol, the alpha 2-adrenergic agonist clonidine and the sulfonylurea tolbutamide lacked effects on [Ca2+]i. In addition to providing support for the concept that glucagon release is positively modulated by [Ca2+]i, the results demonstrate that cAMP raises [Ca2+]i in the alpha 2-cells by mobilizing calcium incorporated in response to glucose.