Glucagon secreted from pancreatic -cells plays a critical role in glycemia, mainly by hepatic glucose mobilization. In diabetic patients, an impaired control of glucagon release can worsen glucose homeostasis. Despite its importance, the mechanisms that regulate its secretion are still poorly understood. Since -cells are particularly sensitive to neural and paracrine factors, in this report we studied the role of purinergic receptors and extracellular ATP, which can be released from nerve terminals and -cell secretory granules. Using immunocytochemistry, we identified in -cells the P2 receptor subtype P2Y₁ as well as the P1 receptors A₁ and A2A. In contrast, only P2Y₁ and A₁ receptors were localized in -cells. To analyze the role of purinergic receptors in alpha-cell function, we studied their participation in Ca²⁺ signaling. At low glucose concentrations, mouse alpha-cells exhibited the characteristic oscillatory Ca²⁺ signals that lead to secretion. Application of ATP (1-10 micromolar) abolished these oscillations or reduced their frequency in alpha-cells within intact islets and isolated in culture. ATP--S, a non-hydrolyzable ATP-derivative, indicated that the ATP effect was mainly direct rather than through ATP-hydrolytic products. Additionally, adenosine (1-10 micromolar) was also found to reduce Ca²⁺ signals. ATP-mediated inhibition of Ca²⁺ signaling was accompanied by a decrease in glucagon release from intact islets in contrast to the adenosine effect. Using pharmacological agonists, we found that only P2Y₁ and A_2A were likely involved in the inhibitory effect on Ca²⁺ signaling. All these findings indicate that extracellular ATP and purinergic stimulation are effective regulators of the alpha-cell function.