Pharmacological studies demonstrated that ATP elevates intracellular calcium ([Ca⁺⁺]_i) in supraoptic nucleus (SON) neurons primarily by activation of P2X2 and P2Y1 purinergic receptors [P2Y1R; (18)]. The current studies provide evidence for the presence of P2Y1R protein in SON neurons, evidence that activation of these P2Y1Rs induces an increase in [Ca⁺⁺]_i from both intracellular stores and Ca⁺⁺ influx, and functional evidence that activation of P2Y1Rs induces vasopressin (VP) and oxytocin (OT) hormone release. Pretreatment of Fura-2 AM loaded explants of the hypothalamo-neurohypophyseal system (HNS) with thapsigargin (TG) significantly (~80%) reduced the increase in [Ca⁺⁺]_i induced by the P2Y1R specific agonist, 2-methylthio-ADP (2-MeSADP). In contrast, the increase in [Ca⁺⁺]_i was slightly (~20%) decreased in calcium free medium. The calcium response to 2-MeSADP was completely blocked by the P2Y1R specific antagonist, MRS2179 or by a combination of TG pretreatment and calcium free medium. It was absent in P2Y1 knockout mice (P2Y1^-/-). 2-MeSADP significantly increased VP and OT release from perifused rat and wild-type mouse HNS explants compared to control. MRS2179 prevented this response in wild-type mouse, but did not prevent ATP-induced hormone release from rat explants. 2-MeSADP did not induce hormone release from P2Y1^-/- explants. These findings support a potential role for P2Y1Rs in regulation of VP and OT release. The finding that P2Y1R activation induces a small Ca⁺⁺ influx suggests that P2Y1Rs may regulate VP release by modifying ion channels such as stretch inactivated cation channels.