To determine whether hepatic urea production is limited at low hepatic O2 delivery (DO2) by O2 itself or by the availability of substrate for urea synthesis, we isolated livers from normal rats and perfused them with Krebs-Henseleit bicarbonate (KHB) buffer, KHB + 5 mM NH4Cl, or KHB + 5 mM glutamine (Gln) as an NH3 donor. The pump flow was lowered in stages, and we determined at each flow rate inflow and outflow O2 content and urea levels in the outflow perfusate. Urea production in Gln-perfused livers remained constant at high DO2 and declined in direct proportion to DO2 below a critical oxygen delivery (DO2crit, the point below which the hepatic O2 consumption [VO2] becomes limited by the hepatic DO2). The DO2crit calculated from the urea release-DO2 relationship (147 +/- 32 microl/min/dry g) was similar to the DO2crit calculated from the VO2-DO2 relationship (158 +/- 26 microl/min/dry g). When Gln concentration and flow rate were maintained constant while decreasing PO2 in the inflow perfusate (as well as hepatic DO2), urea production declined below the DO2crit. Furthermore, when Gln concentration in the perfusate was gradually reduced while keeping hepatic DO2 constant, urea production decreased proportionally with Gln concentrations in the perfusate. Consequently, urea production is dependent on Gln and O2 availability and becomes limited at the same DO2crit determined by the VO2-DO2 relationship.