Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd³⁺ as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl⁻ permeability and stimulates cell volume recovery through activation of P₂ receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd³⁺(200 μM) inhibited volume-sensitive ATP release by >90% (P < 0.001), inhibited cell volume recovery from swelling (P < 0.01), and uncoupled cell volume from increases in membrane Cl⁻ permeability (P < 0.01). Moreover, Gd³⁺ had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd³⁺ might prove to be an effective inhibitor of ATP-permeable channels once they are identified.