Vasopressin release from the hypothalamo-neurohypophyseal system (HNS) is stimulated by ATP activation of P_2X purinergic receptors and by activation of alpha1-adrenergic receptors by phenylephrine (PE). These responses are potentiated by simultaneous exposure to ATP+PE. Potentiation was blocked by depleting intracellular calcium stores with thapsigargin. To test the hypothesis that the synergistic response to ATP+PE reflects alterations in the intracellular calcium concentration ([Ca⁺⁺]_i), [Ca⁺⁺]_i was monitored in supraoptic neurons in HNS explants loaded with Fura 2-AM. Both ATP and PE induced rapid, but transient elevations in [Ca⁺⁺]_i. In 0.3 mM Ca⁺⁺, the peak response to ATP was greater than to PE, but did not differ from the peak response to ATP+PE. A sustained elevation in [Ca⁺⁺]_i was induced by ATP+PE, that was greater than ATP or PE alone. In 2 mM Ca⁺⁺, the peak response to ATP+PE was significantly greater than to either ATP or PE alone, and the sustained response to ATP+PE was greater than to either agent alone. Responses were comparable in the presence of tetrodotoxin. The sustained elevation in [Ca⁺⁺]_i was also observed when ATP+PE was removed after 1 min, but it was eliminated by either thapsigargin or removing external calcium indicating that both calcium influx and calcium release from internal stores are required. Some cells were vasopressinergic based on a VP-induced increase in [Ca⁺⁺]_i. These observations support the hypothesis that simultaneous exposure to ATP+PE induces a different pattern of [Ca⁺⁺]_i than either agent alone that may initiate events leading to synergistic stimulation of VP release.