BACKGROUND/AIMS: Purinergic signaling potentially contributes to many liver functions. Therefore, the purpose of these studies was to characterize adenosine 5'-triphosphate (ATP) release from human hepatocytes, and to determine the role of extracellular ATP in the autocrine regulation of Cl- permeability and cell volume homeostasis. METHODS: Release of ATP (luciferase-luciferin assay), Cl- currents (whole-cell patch clamp), and cell volume (Coulter Multisizer) were measured in human hepatocytes within 12 h of isolation. RESULTS: Hepatocyte swelling increased bioluminescence from basal values of 11.21+/-0.45 to 178.29+/-44.49 and 492.15+/-89.41 arbitrary light units following 20 and 40% buffer dilutions, respectively (p<0.001), representing an increase in extracellular ATP from approximately 10 to >300 nM. Whole-cell Cl- currents activated during exposure to hypotonic buffer (15% less mosmol, 126.34+/-36.49 pA/pF) and ATP (10 microM, 71.92+/-15.48 pA/pF) exhibited outward rectification, time-dependent inactivation at depolarizing potentials, and sensitivity to the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Removal of extracellular ATP (apyrase) prevented volume-sensitive current activation. Exposure to hypotonic buffer (30% less mosmol) increased mean relative volume to 1.092+/-0.004 by 2.5 min, and volume recovery (1.019+/-0.002 by 30 min) was abolished by NPPB, apyrase, and the P2 receptor antagonist suramin. CONCLUSIONS: These findings indicate that human hepatocytes exhibit constitutive and volume-dependent ATP release, which is a critical determinant of membrane Cl- permeability and cell volume regulation. ATP release may represent an extracellular signaling pathway that couples the cellular hydration state to important hepatic functions.