Hepatic uptake of five common bile acids was examined in isolated rat hepatocytes. Taurocholic acid (TCA), glycocholic acid (GCA), cholic acid (CA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) were studied. Uptake was extremely rapid and was linear for at least 45 sec for all bile acids tested at substrate concentrations from 1 to 400 micrometers. Both nonsaturable binding to the cell surface membrane and the initial rate of uptake (V0) of the dihydroxy bile acids (DCA and CDCA) were about ten times greater than those of the trihydroxy bile acids (TCA, GCA and CA) which correlates with the higher lipophilicity of the dihydroxy bile acids. Kinetic analysis demonstrated that uptake of these bile acids was due both to a saturable process and a linear process. The apparent diffusion constant (Dapp) of the unsaturable process for the dihydroxy bile acids was also ten times greater than that for the trihydroxy bile acids. After correction for the nonsaturable binding to the cell membrane and linear entry, the Km and Vmax for the uptake was determined. Conjugation with taurine decreased the Km of CA but not the Vmax, while glycine conjugation did not alter either parameter, suggesting that conjugation with taurine may increase its affinity for the transport system. The trihydroxy bile acids have a higher affinity but a lower transporting capacity for the saturable process than the dihydroxy bile acids. In vivo hepatic extraction appears to be more dependent on the affinity of the bile acid for the transport system than the capacity at which it can be transported.