ATP increases intracellular calcium ([Ca⁺⁺]_i) in supraoptic nucleus (SON) neurons in hypothalamo-neurohypophyseal system (HNS) explants loaded with the Ca⁺⁺-sensitive dye, Fura-2 AM. Involvement of P2X purinergic receptors (P2XR) in this response was anticipated, because ATP stimulation of vasopressin (VP) release from HNS explants required activation of P2XRs, and activation of P2XRs induced an increase in [Ca⁺⁺]_i in dissociated SON neurons. However, the ATP-induced increase in [Ca⁺⁺]_i persisted after removal of Ca⁺⁺ from the perifusate ([Ca⁺⁺]_o). This suggested involvement of P2YRs, because P2YRs induce Ca++ release from intracellular stores while P2XRs are Ca⁺⁺ permeable ion channels. Depletion of [Ca⁺⁺]_i stores with thapsigargin (TG) prevented the ATP-induced increase in [Ca⁺⁺]_i in zero, but not in 2mM [Ca⁺⁺]_o indicating that both Ca⁺⁺ influx and release of intracellular Ca⁺⁺ contribute to the ATP response. Ca⁺⁺ influx was partially blocked by cadmium indicating a contribution of voltage-gated Ca⁺⁺ channels. PPADS (pyridoxal-phosphate-6-azophenyl-2', 4'-disulphonic acid), and isoPPADS, P2XR antagonists, attenuated, but didn't abolish the ATP-induced increase in [Ca⁺⁺]_i. Combined treatment with PPADS or isoPPADS and TG prevented the response. A cocktail of P2YR agonists consisting of UTP, UDP, and 2-MethylS-ADP increased [Ca⁺⁺]_i (with or without tetrodotoxin) that was markedly attenuated by TG. 2-MethylSADP alone induced consistent and larger increases in [Ca⁺⁺]_i than UTP or UDP. MRS2179, a specific P2Y1R antagonist, eliminated the response to ATP in zero [Ca⁺⁺]_o. Thus, both P2X and P2Y receptors participate in the ATP-induced increase in [Ca⁺⁺]_i, and the P2Y1R subtype is more prominent than P2Y2, P2Y4 , or P2Y6 receptors in SON.