We have investigated the effects of extracellular (pHo) and intracellular (pHi) pH on membrane potential difference (PD) and cell conductance (gcell) in rat hepatocytes in primary culture. PD and pHi were measured continuously by using intracellular microelectrodes and the pH-sensitive fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxy fluorescein (BCECF), respectively, during abrupt changes in the pHo or ionic composition of extracellular perfusate. In the presence of 25 mM HCO3-, PD, gcell, and pHi averaged (+/- SE) -32 +/- 1 mV, 16.4 +/- 1.0 nS, and 7.32 +/- 0.01, respectively. The transference number for K+ (tk+), which reflects the fractional contribution of K+ conductance to gcell, averaged 0.36 +/- 0.03. Exposure to 1 mM Ba2+ produced membrane depolarization and decreased tK+ by approximately 90%. Lowering pHo by a variety of maneuvers in the presence and absence of HCO3- consistently decreased pHi, decreased gcell (approximately 30 nS per unit change in pHi), and depolarized PD. Increasing pHo had opposite effects, but the changes in gcell were generally greater with intracellular acidification than alkalinization. The decrease in PD produced by lowering pHo was associated with a decrease in tK+ of 73 +/- 2% and was inhibited by Ba2+. Exposure to butyrate or withdrawal of NH+4, which lowered pHi without changing pHo, also caused depolarization of PD and a decrease in gcell that was inhibited by Ba2+. These observations indicate that the PD of hepatocytes is strongly influenced by pHi, with or without changes in pHo, and they further suggest that the effects of pH on PD are mediated through changes in plasma membrane K+ conductance.