The impulse-response technique was used to investigate the influence of changes in the perfusate concentration of human serum albumin (HSA; 1.5-25 g/L) on the distribution and elimination kinetics of [14C]diclofenac in the isolated perfused rat liver. Output data were analyzed by a linear systems approach in combination with the axial dispersion model of hepatic elimination. This stochastic model is characterized by a dimensionless parameter (the dispersion number, DN) that quantifies the relative spreading of a substance as it passes through the liver. The two-compartment form of the axial dispersion model, which assumes that the radial transfer of a substance between the vascular and cellular spaces proceeds at a finite rate, was used to describe the output profiles for diclofenac, thereby providing estimates for DN and the first-order rate constants for the transfer of drug between the vascular and cellular compartments (k12 and k21) and its sequestration from the cellular compartment (fc8l). With a change in perfusate HSA concentration, the only one of these parameters to alter significantly (analysis of variance, p < 0.05) was the uptake rate constant (k12), which increased from 0.091 ± 0.016 (mean ± standard deviation) to 0.79 ± 0.09s-1 as HSA decreased from 25 to 1.5 g/L. Most of this change could be accounted for by an increase in the fraction of diclofenac unbound in perfusate, from 0.0030 to 0.0407 as HSA decreased from 25 to 1.5 g/L. The relative magnitudes of k12, k21, and ke, suggest that although membrane permeability has an Important bearing on the distribution kinetics of diclofenac within the liver and therefore the shape of the output profile following an impulse input, the elimination of diclofenac is not subject to a hepatocyte membrane permeability limitation. The DN for diclofenac (0.3 to 0.4) was independent of protein concentration and was similar in magnitude to that determined for the noneliminated reference marker albumin. These results suggest that under conditions of high binding within the perfusion medium, the axial spreading of diclofenac within the liver is determined primarily by the heterogeneity of hepatic blood flow.