We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aik, aCliand aNai), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 μml-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 m m tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33±1 mV. l-alanine uptake first depolarized Vm by 2±0.2 mV and then hyperpolarized Vm by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aNaiincreased by 30% from 19±2 to 25±3 m m (P < 0.01), and aKidid not change significantly from 83±3 m m. However, with added ouabain (1 m m) l-alanine caused only a 2-mV increase in Vm, but now aKidecreased from 61±3 to 54±5 m m (P < 0.05). Hyperpolarization of Vm by l-alanine uptake also resulted in a 38% decrease of aClifrom 20±2 to 12±3 m m (P < 0.001). Changes in Vm and VCl - Vm voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes. Added Ba2+ abolished the l-alanineinduced hyperpolarization, and aCliremained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte aKiis regulated by increased Na+-K+ pump activity during l-alanine uptake in spite of cell swelling and increased Vm due to increased K+ conductance. The hyperpolarization of Vm during l-alanine uptake provides electromotive force to decrease aCli. The latter may contribute to hepatocyte volume regulation during organic solute transport.