The mechanisms by which hypoglycemia stimulates glucagon release are still poorly understood. In particular, the relative importance of direct metabolic coupling versus paracrine regulation by beta-cell secretory products is unresolved. Here, we compare the responses to glucose of 1) alpha-cells within the intact mouse islet, 2) dissociated alpha-cells, and 3) clonal alphaTC1-9 cells. Free cytosolic concentrations of ATP ([ATP](c)) or Ca(2+) ([Ca(2+)](c)) were imaged using alpha-cell-targeted firefly luciferase or a green fluorescent protein-based Ca(2+) probe ("pericam"), respectively. Consistent with a direct effect of glucose on alpha-cell oxidative metabolism, an increase in glucose concentration (from 0 or 3 mmol/l to 20 mmol/l) increased [ATP](c) by 7-9% in alpha-cells within the intact islet and by approximately 4% in alphaTC1-9 cells. Moreover, glucose also dose-dependently decreased the frequency of [Ca(2+)](c) oscillations in both dissociated alpha-cells and alphaTC1-9 cells. Although the effects of glucose were mimicked by exogenous insulin, they were preserved when insulin signaling was blocked with wortmannin. Addition of ZnCl(2) slightly increased the frequency of [Ca(2+)](c) oscillations but failed to affect glucagon release from either islets or alphaTC1-9 cells under most conditions. We conclude that glucose and insulin, but not Zn(2+) ions, independently suppress glucagon secretion in the mouse.