Hepatocellular Cl^- flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl^- current (VRAC) has been well described in hepatocytes, the Cl^- channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes, WIF-B and HEK293T cells were measured under patch electrode voltage clamp. With K⁺-free salts of Cl^- comprising the major ions externally and internally, hyperpolarizing steps between -40 and -140mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half maximal at - 63mV and 28-38% of maximum at -30 to -45mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl^-, shifting close to 58mV per 10- fold change in Cl^- concentration. Time-dependent inward Cl^- currents and a ClC-2- specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. 4,4'- diisothiocyanatostilbene-2,2'-disulphonic acid (0.1mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the carboxyl-terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl- channel that could provide a significant depolarizing influence in the hepatocyte.