The hepatic uptake of two sulfonylureas, glisoxepide and glibenclamide, was investigated in isolated rat hepatocytes. Two transport processes were defined: passive physical diffusion and saturable carrier transport. For diffusion at pH 7.4 the permeability coefficients were 3.3 x 10(-6) cm/s for glisoxepide and 10.6 x 10(-6) cm/s for glibenclamide. Saturable uptake differed among the sulfonylureas. Glibenclamide uptake was neither energy- nor sodium-dependent and temperature dependence was linear. The apparent activation energy for saturable glibenclamide uptake was 15.2 kJ/mol and Q10 values for uptake between 7 and 37 degrees C were 1.17 +/- 0.12. Saturable glibenclamide uptake exhibited a Km = 3.1 microM and a Vmax = 416 pmol/mg cell protein per min. Thus glibenclamide uptake was defined kinetically as a facilitated diffusion process. Glisoxepide uptake revealed two Km values: Km1 = 2-3 microM and Vmax1 = 200 pmol/mg protein per min, and Km2 = 110 microM and Vmax2 = 1600 pmol/mg protein per min. Uptake at low and high substrate concentration was energy-dependent, sodium-dependent and was inhibited by ouabain. Temperature dependence increased markedly beyond 22 degrees C and the apparent activation energy was 59.7 kJ/mol at low Km1 glisoxepide concentrations and 60.3 kJ/mol at high Km2 concentrations. Whereas glisoxepide was slowly taken up into AS-30D hepatoma cells, glibenclamide was not. The hepatic uptake of glibenclamide was not inhibited by glisoxepide but glibenclamide inhibited glisoxepide uptake. The inhibition by glibenclamide was noncompetitive. Isolated hepatocytes accumulated the sulfonylureas markedly and metabolized both. The metabolized radioligands were slowly released into the incubation buffer. The results indicate that the hepatic uptake of the two sulfonylureas is by carrier-mediated transport. The uptake processes are, however, strikingly different, indicating heterogeneity of sulfonylurea transporters.