Primary cultures and plasma membrane vesicles were used to characterize Na+ and HCO3- transport by rat hepatocytes. Na+ uptake into hepatocytes was stimulated ~ 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 ~ 10-fold by an in > out HCO3- concentration gradient. 22Na efflux was also increased by valinomycin and an in > out K+ concentration gradient in the presence but not absence of HOC3-. Intracellular pH (pH(i)) 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 P(CO2). 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 > out K+ concentration gradient caused pH(i) 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 > 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.