A stable intracellular ionic environment is necessary for hepatocytes to function normally. Thus, during hypotonic shock or L-alanine uptake, hepatocytes swell and then exhibit a regulatory volume decrease (RVD), which comprises an increase in K\sp+ conductance (G\sb{\rm K}), an increased K\sp+ efflux, and a hyperpolarization of transmembrane potential (V\sb{\rm m}). Since hepatocyte intracellular Cl\sp- has been demonstrated to distribute passively with V\sb{\rm m}, this study is designed to test the hypothesis that the hypotonic shock- or L-alanine uptake-induced hyperpolarization of V\sb{\rm m} might provide an electromotive force for the efflux of hepatocyte intracellular Cl\sp-, which in turn would contribute osmotically to the RVD in hepatocytes. Double-barreled ion-selective microelectrodes were used to measure the changes of hepatocyte transmembrane potential, intracellular ionic activities (especially intracellular Cl\sp- activity, (a\sp{\rm i}\sb{\rm Cl})), and intracellular water volume during either anisotonic stress or L-alanine uptake. Hepatocyte V\sb{\rm m} hyperpolarized, (a\sp{\rm i}\sb{\rm Cl}) decreased, intracellular K\sp+ activity (a\sp{\rm i}\sb{\rm K}) decreased, and intracellular water volume increased during hyposmotic stress. When perfused with L-alanine, hepatocyte V\sb{\rm m} exhibited a transient depolarization followed by repolarization and then underwent a constant hyperpolarization. Meanwhile, hepatocyte intracellular Na\sp+ activity (a\sp{\rm i}\sb{\rm Na}) increased, a\sp{\rm i}\sb{\rm K} & a\sp{\rm i}\sb{\rm Cl} decreased, and intracellular water volume increased. In both hypotonic shock and L-alanine uptake conditions, the decreased a\sp{\rm i}\sb{\rm K} could be attributed to cell swelling. However, the decrease in a\sp{\rm i}\sb{\rm Cl} was greater than could be accounted for by cell swelling. When the change of V\sb{\rm m} was inhibited by K\sp+ channel blockers, the change of a\sp{\rm i}\sb{\rm Cl} was also inhibited. Based on the measured a\sp{\rm i}\sb{\rm Cl}, Cl\sp- was always at its electrochemical equilibrium in all of the control and experimental conditions. The conclusions of this study emphasize the passive distribution of hepatocyte intracellular Cl\sp- with the changes of V\sb{\rm m} induced by hypotonic stress and L-alanine uptake. Thus, the data strongly support the idea that the hypotonic shock- or L-alanine uptake-induced hyperpolarization of V\sb{\rm m} provides electromotive force for the efflux of hepatocyte intracellular Cl\sp-. This could contribute to hepatocyte volume regulation during both hypotonic shock and organic solute transport
