The dependence of gluconeogenesis and O2 uptake on PO2 in isolated rat hepatocytes is presented. Maintenance of steady-state PO2 was achieved with an oxystat system (Biochem. J. 236: 765-769, 1986). O2 uptake showed a half-maximal (K0.5) value of 0.5 Torr PO2, whereas the glucose synthesis rate was half-maximal at 1.2 Torr PO2. Halothane at concentrations greater than 1 mM exerted a parallel inhibition of O2 uptake and glucose synthesis at all PO2 levels studied. In contrast, at halothane concentrations less than 1 mM, inhibition of glucose synthesis occurred only at less than 20 Torr PO2. At these low concentrations, halothane was without significant effects on cellular O2 uptake. In isolated mitochondria, inhibition of O2 uptake was already half-maximal at a halothane concentration of 0.5 mM. In this subcellular system the inhibitory effect of halothane was independent of PO2. These results demonstrate that the critical PO2 at which cellular O2 utilization begins to decrease and the PO2 at which glucose synthesis begins to decrease are comparable; both PO2 levels are approximately 5 Torr. The metabolic zonation of the liver lobule is discussed in view of the results presented.