Hepatocytes isolated from 10 Dorset wethers that were treated with excipient or 1.0 g/d of phlorizin for 72 h were used to determine the effects of increased glucose demand on utilization of [1-(14)C]propionate and [1-(14)C] alanine for oxidative metabolism and gluconeogenesis. Control and phlorizin-treated wethers excreted 0 and 62.8 g/d of glucose into the urine, respectively. Phlorizin treatment tended to increase conversion of propionate and alanine to CO2. A phlorizin x substrate interaction for conversion to glucose indicated that conversion of alanine to glucose was increased more by phlorizin treatment than was conversion of propionate (285 vs 166% of controls). Phlorizin treatment did not affect estimated Ks for conversion of substrates to either CO2 or glucose; however, phlorizin increased estimated Vmax for conversion of substrates to CO2 and tended to increase estimated Vmax for conversion of substrates to glucose. Phlorizin treatment slightly increased the ratio of conversion of propionate to glucose compared with CO2 and slightly decreased the ratio of conversion of alanine to glucose compared with CO2. In vitro addition of 2.5 mM NH4Cl decreased conversion of propionate to CO2 and glucose but had little effect on conversion of alanine to CO2 and glucose. Estimated Ks and Vmax for conversion of substrates to CO2, Ks for conversion of substrates to glucose, and Vmax for conversion of alanine to glucose were not affected by NH4Cl; however, Vmax for conversion of propionate to glucose was decreased by NH4Cl. These data indicate that although utilization of propionate for gluconeogenesis is extensive, amino acids have the potential to increase in importance as gluconeogenic substrates when glucose demand is increased substantially. Furthermore, excess ammonia decreases the capacity of hepatocytes to utilize propionate for oxidation and gluconeogenesis.