P rimary cultures and plasma membrane vesicles were used to characterize Na+ and HCO3- transport by rat hepatocytes. Na+ uptake into hepatocytes was stimulated approximately 10-fold by 25 mM extracellular HCO3-.HCO3--stimulated Na+ uptake was saturable, abolished by 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid stilbene (SITS), and unaffected by amiloride or Cl- removal. Neither propionate nor acetate reproduced this effect of HCO3-. 22Na efflux from preloaded hepatocytes was similarly increased approximately 10-fold by an in greater than out HCO3- concentration gradient. 22Na efflux was also increased by valinomycin and an in greater than out K+ concentration gradient in the presence but not absence of HCO3-. Intracellular pH (pHi) measured with the pH-sensitive fluorochrome 2',7'-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) decreased at a rate of 0.227 (+/- 0.074 SEM) pH units/min when extracellular HCO3- concentration was lowered from 25 to 5 mM at constant PCO2. This intracellular acidification rate was decreased 50-60% in the absence of Na+ or presence of SITS, and was unaffected by amiloride or Cl- removal. Membrane hyperpolarization produced by valinomycin and an in greater than out K+ concentration gradient caused pHi to fall; the rate of fall was decreased 50-70% by Na+ removal or SITS, but not amiloride. An inside positive K+ diffusion potential and a simultaneous out greater than in HCO3- gradient produced a transient 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS) sensitive, amiloride-insensitive 22Na accumulation in basolateral but not canalicular membrane vesicles. Rat hepatocytes thus exhibit electrogenic basolateral Na+/HCO3- cotransport.