Ionotrophic purinergic (P2X) receptors function as receptor-gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na ุ and Ca 2ุ . Based on evidence that extracellular adenosine 5-triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP-dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 23-O-(4-benzoyl-benzoyl)-adenosine 5-triphosphate (BzATP; 10 M) caused a rapid increase in membrane Na ุ conductance. Similarly, with Fluo-3 fluorescence, BzATP induced an increase in intracellular [Ca 2ุ ]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu 2ุ , and the P2X4 modulators Zn 2ุ and ivermectin (0.3-3 M) each increased intracellular [Ca 2ุ ]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen-rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na ุ and Ca 2ุ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism.