Hepatic distributional volumes were investigated in the in situ perfused rat liver. Perfusion experiments were conducted using Krebs bicarbonate buffer delivered via the portal vein in single-pass mode at a total flow rate of 15 mL/min. A bolus dose of normal erythrocytes (RBC, vascular marker) and Evans blue (EB, extracellular marker) respectively was administered in the presence and absence of protein. At the end of the experiment, liver total water content was determined by desiccation and freeze-drying methods. Similar moment analysis results and superimposable effluent curves were obtained in the presence (RBC, mean transit time [MM: 7.31 0.45 s and volume of distribution [V]: 0. 17 0.01 mL/g; EB, WE 10.9 +/- 0.62 s and V: 0.25 +/- 0.02 mL/g) and in the absence (RBC, MTT. 7.55 +/- 0.84 s and V. 0.18 +/- 0.02 mL/g; EB, MTT: 9.24 +/- 0.77 s and V: 0.20 +/- 0.02 mL/g) of protein, which indicates that the hepatic distribution of RBC and EB within the liver is not influenced by protein. Furthermore, the almost identical results obtained with the desiccation and freeze-drying methods clearly suggest that the freeze-drying method can be used as an alternative to desiccation for the estimation of liver water content.