Futile cycling between 4-methylumbelliferone and its sulfate and glucuronide conjugates was examined in the single-pass perfused rat liver preparation. The steady-state hepatic extraction ratio of 4-methylumbelliferone was found to be high (0.97) at a low input concentration of 0.005 μmol/L (tracer), with a net 4-methylumbelliferyl sulfate/4-methylumbelliferyl glucuronide ratio of about 5:1; at 63 μmol/L the steady-state extraction ratio had remained constant despite a shift from net sulfation to net glucuronidation. At higher input 4-methylumbelliferone concentrations, saturation was evidenced by a decreased steady-state extraction ratio and reduced net sulfation and net glucuronidation. Because 4-methylumbelliferyl sulfate and 4-methylumbelliferyl glucuronide deconjugation would result in an intracellular accumulation of 4-methylumbelliferone, the phenomenon was monitored with a shift in tracer [3H]4-methylumbelliferone metabolism from sulfation to glucuronidation with increased intracellular 4-methylumbelliferone concentration. When 4-methylumbelliferyl sulfate (0 to 890 μmol/L) or 4-methylumbelliferyl glucuronide (0 to 460 μmol/L) was delivered simultaneously with tracer [3H]4-methylumbelliferone to the rat liver, notable desulfation of 4-methylumbelliferyl sulfate (18% to 38% rate in) but little deglucuronidation of 4-methylumbelliferyl glucuronide (1.2% to 2.1% rate in) was observed. With 4-methylumbelliferyl sulfate, 4-methylumbelliferone and 4-methylumbelliferyl glucuronide were readily found as metabolites, whereas with 4-methylumbelliferyl glucuronide, levels of the metabolites, 4-methylumbelliferone and 4-methylumbelliferyl sulfate, were much reduced. 4-Methylumbelliferyl sulfate and not 4-methylumbelliferyl glucuronide shifted tracer [3H]4-methylumbelliferone metabolism from [3H]4-methylumbelliferyl sulfate to [3H]4-methylumbelliferyl glucuronide formation in a concentration-dependent fashion. The steady-state ex traction ratio for 4-methylumbelliferyl sulfate (0.1 to 0.3) was comparatively higher than that for 4-methylumbelliferyl glucuronide (0.05), and it was found to increase with concentration, an observation explained by the nonlinear protein binding of 4-methylumbelliferyl sulfate. Biliary excretion rates for 4-methylumbelliferone and 4-methylumbelliferyl sulfate were proportional to their input or net formation rates, regardless of whether 4-methylumbelliferyl erone, 4-methylumbelliferyl glucuronide or 4-methylumbelliferyl sulfate was administered. By contrast, the excretion rate of 4-methylumbelliferyl glucuronide when administered was only 1/25 the excretion of 4-methylumbelliferyl glucuronide formed from 4-methylumbelliferone and 4-methylumbelliferyl sulfate. The extent of choleresis paralleled the excretion patterns of preformed and formed 4-methylumbelliferyl glucuronide; bile flow was normal with 4-methylumbelliferyl glucuronide administration and was markedly enhanced with increased 4-methylumbelliferone or 4-methylumbelliferyl sulfate administration. The data suggest the presence of a transmembrane barrier for entry of 4-methylumbelliferyl glucuronide and not 4-methylumbelliferyl sulfate or 4-methylumbelliferone into hepatocytes. On utilization of a tubular-flow model to describe the data obtained for 4-methylumbelliferyl sulfate, 4-methylumbelliferone and 4-methylumbelliferyl glucuronide, we were able to attribute the effects of protein binding, conjugation and deconjugation in explaining the observations. The simulations substantiated the notion of a barrier for 4-methylumbelliferyl glucuronide entry into hepatocytes and a limitation caused by protein binding for 4-methylumbelliferyl sulfate removal at low input concentrations. The simulations further showed that desulfation, if absent, increased the total sulfation rate (15%), decreased very slightly the net glucuronidation rate of 4-methylumbelliferone and increased the overall steady-state extraction ratio. In absence of deglucuronidation, the steady-state extraction ratio and net glucuronidation rate were very slightly increased (3%) without perturbing the net sulfation rate. Futile cycling between a precursor and product results in a reduction of net appearance of that product in the venous circulation or bile and conduces to increasing the formation of other metabolites that are not involved in interconversion. The outcome is highly dependent on protein binding and transmembrane characteristics of the species and the enzymatic parameters for the forward and backward reactions. (HEPATOLOGY 1993;17:838–853.)