Anterior pituitary cells express cation-conducting P2X receptor channels (P2XRs), but their molecular identity, electrophysiological properties, cell-specific expression pattern, and physiological roles have been only partially characterized. In this study, we show by quantitative RT-PCR that mRNA transcripts for the P2X4 subunit are the most abundant in rat anterior pituitary tissue and confirm the P2X4R protein expression by Western blot analysis. Single-cell patch-clamp recordings show that extracellular ATP induced an inward depolarizing current in a majority of thyrotropin-releasing hormone-responsive pituitary cells, which resembled the current profile generated by recombinant P2X4R. The channels were activated and desensitized in a dose-dependent manner and deactivated rapidly. Activation of these channels led to stimulation of electrical activity and promotion of voltage-gated and voltageinsensitive Ca 2ϩ influx. In the presence of ivermectin, a specific allosteric modulator of P2X4Rs, there was an approximately fourfold increase in the maximum amplitude of the ATP-induced inward current, accompanied by an increase in the sensitivity of receptors for ATP, slowed deactivation of receptors, and enhanced ATP-induced prolactin release. These results indicate that thyrotropin-releasing hormone-responsive cells, including lactotrophs, express homomeric and/or heteromeric P2X4Rs, which facilitate Ca 2ϩ influx and hormone secretion. adenosine 5=-triphosphatase; adenosine 5=-triphosphatase-gated receptor channels; calcium; lactotrophs; prolactin; ivermectin IN ADDITION TO ITS INTRACELLULAR FUNCTIONS, ATP can be released by excitable and nonexcitable cells and act as an extracellular messenger through the stimulation of P2Y receptors (P2YRs) and P2X receptors (P2XRs). To date, eight mammalian P2YRs have been identified and are denoted as