Binding of prednisolone to rabbit tissue was determined experimentally and by computation to assess factors and proteins responsible for tissue distribution of this corticosteroid. Binding in tissue slices ranged from 36% in the lung to 83% in the liver. The results obtained were compared with steady-state tissue distribution data generated in vivo in the rabbit. Linear uptake with good agreement between the two sets of data was observed for the skeletal muscle and kidney, but not for the liver and lung. In vivo uptake of prednisolone in the liver is severely affected by metabolism and biliary excretion, while in vitro distribution into liver slices showed a pattern typical of saturation of a low-capacity site followed by linear nonspecific binding at higher concentrations. Specific saturable binding (K1 = 1.95 X 10(7) M-1) accounted for 74% of drug bound at a steroid concentration in the liver of 0.3 X 10(-6) M, but its contribution decreased to 5% at a concentration of 20 X 10(-6) M. Expected prednisolone binding in rabbit tissue was calculated based on various effective protein concentrations in tissues and binding parameters of prednisolone to rabbit plasma. The use of total Lowry protein concentrations markedly overestimates, while albumin (and transcortin) concentrations severely underestimate the tissue binding of prednisolone. The thiopental effective protein fraction best approximates the in vivo and tissue slice binding of prednisolone. Metabolism and saturable binding in liver complicate the otherwise relatively linear uptake of prednisolone into various tissues where macromolecules other than albumin dominate in tissue binding of the steroid.