Glucagon is released from α-cells present in intact pancreatic islets at glucose concentrations below 4mM, whereas higher glucose levels inhibit its secretion. The mechanisms underlying the suppression of α-cell secretory activity are poorly understood, but two general types of models have been proposed as follows: direct inhibition by glucose or paracrine inhibition from non-α-cells within the islet of Langerhans. To identify α-cells for analysis, we utilized transgenic mice expressing fluorescent proteins targeted specifically to these cells. Measurements of glucagon secretion from pure populations of flow-sorted α-cells show that contrary to its effect on intact islets, glucose does stimulate glucagon secretion from isolated α-cells. This observation argues against a direct inhibition of glucagon secretion by glucose and supports the paracrine inhibition model. Imaging of cellular metabolism by two-photon excitation of NAD(P)H autofluorescence indicates that glucose is metabolized in α-cells and that glucokinase is the likely rate-limiting step in this process. Imaging calcium dynamics of α-cells in intact islets reveals that inhibiting concentrations of glucose increase the intracellular calcium concentration and the frequency of α-cell calcium oscillations. Application of candidate paracrine inhibitors leads to reduced glucagon secretion but did not decrease the α-cell calcium activity. Taken together, the data suggest that suppression occurs downstream from α-cell calcium signaling, presumably at the level of vesicle trafficking or exocytotic machinery.