The hepatocytic entry of acetaminophen sulfate conjugate was examined in the rat liver, perfused with red cells with and without albumin, by use of the multiple-indicator dilution technique. [3H]acetaminophen sulfate was injected into the portal vein in a bolus of blood containing 51Cr-labeled red blood cells (a vascular reference), sucrose (a low-molecular-weight interstitial reference) or 125I-labeled albumin (a high-molecular-weight interstitial reference, included when albumin was present), and the time courses of their outflow into the hepatic venous blood were observed. The [3H]acetaminophen sulfate, which binds partially to albumin, emerged between albumin and sucrose in the presence of albumin, processed the upslope of the sucrose curve and showed a late low-in-magnitude tailing; the precession disappeared in the absence of albumin. Biliary excretion of [3H]acetaminophen sulfate was less than 1% of the dose. Quantitative evaluation with a barrier-limited, space-distributed variable transit time model (including rapidly equilibrating albumin binding) accounted for the albumin effect on [3H]acetaminophen sulfate behavior and demonstrated a low liver cell permeability for the acetaminophen sulfate and a small interstitial binding space for its nonalbumin-bound fraction in excess of that for sucrose, which in the absence of albumin was of similar dimensions.