The single-pass perfused rat liver preparation was used to assess the influence of binding to human serum albumin on the steady-state hepatic extraction of diclofenac (n = 8). In the absence of binding protein, the extraction ratio of diclofenac approached unity (range, 0.975-0.992), such that its clearance was perfusion-rate limited. As the binding of diclofenac to protein was increased by the addition of human serum albumin to the perfusion medium, its extraction ratio decreased dramatically, and clearance eventually became capacity limited. The relationship between diclofenac availability and fraction unbound was analyzed with various physiologic models of hepatic drug clearance. The dispersion model, which contains a parameter (the dispersion number) that quantifies the axial spreading of a substrate as it passes along the liver length, provided a significantly better description of the data (p < 0.05) than the undistributed parallel-tube model, which assumes that an eliminated substrate travels through the liver as an undispersed plug, and the well stirred (venous equilibrium) model, which assumes that substrate undergoes infinite mixing as soon as it enters the liver. The dispersion number estimated for diclofenac (mean, 3.03; range, 0.89-7.56) was significantly greater than that predicted from considerations of the transverse heterogeneity of blood flow within the hepatic sinusoidal bed, suggesting that additional factors influenced the relationship between availability and fraction unbound for this compound. Such factors may include transverse heterogeneity of the metabolizing enzyme system(s), axial flux of substrate created by diffusion within hepatic tissue, and protein-facilitated transfer of substrate across an unstirred fluid layer adjacent to the hepatocyte surface.