Purine nucleotides and their analogs increase insulin secretion through activation of pancreatic β-cell P2Y receptors. The present study aimed at determining the role of glucose metabolism in the response to P2Y agonists and whether ATP-activated K⁺ channels (K_ATP channels) are involved in this response. The experiments were performed in the rat isolated pancreas, perfused with a Krebs-bicarbonate buffer supplemented with 2 g/l bovine serum albumin under dynamic glucose conditions from 5 mmol/l baseline to 11 mmol/l. ADPβS (0.5 μmol/l) was selected as a stable and selective P2Y agonist. This compound, ineffective on the 5 mmol/l glucose background, induced a significant threefold increase in insulin release triggered by the glucose challenge. The effect of ADPβS was markedly reduced (P < 0.001) in the presence of an inhibitor of glucose metabolism. In addition to glucose, the ADP analog also amplified the β-cell insulin response to 15 mmol/l methyl pyruvate (P < 0.05), but it was ineffective on the insulin response to 2.5 mmol/l methyl succinate. A nonmetabolic stimulus was applied using tolbutamide (185 μmol/l). Insulin secretion induced by the K_ATP channel blocker was strongly reinforced by ADPβS (P < 0.001), which prompted us to check a possible interplay of K_ATP channels in the effect of ADPβS. In the presence of diazoxide 250 μmol/l and 21 mmol/l KCl, ADPβS still amplified the second phase of glucose-induced insulin secretion (P < 0.001). We conclude that P2Y receptor activation is able to promote insulin secretion through a mechanism, involving β-cell metabolism and a rise in intracellular calcium; this effect does not result from a direct inhibitory effect on K_ATP channels.