NADPH supply for oxidized glutathione (GSSG) reduction was studied in hepatocytes under different steady-state O2 concentrations with controlled infusions of diamide, a thiol oxidant. When bis-chloro-nitrosourea (BCNU) was used to inhibit GSSG reductase, the rate of GSH depletion approximated the rate of diamide infusion, showing that diamide reacted preferentially with GSH under these experimental conditions. Under aerobic conditions without BCNU treatment, the GSH and NADPH pools were largely unaffected and little diamide accumulation or protein thiol oxidation occurred with diamide infusion rates up to 5.3 nmol/10(6) cells per min. However, at greater infusion rates, GSH and NADPH decreased, diamide and GSSG concentrations increased, and protein thiols were oxidized. This critical infusion rate was easily discernible and provided a convenient means to assess the capacity of cells to reduce GSSG as a function of O2 concentration. As the O2 concentration was decreased below 15 microM, the critical infusion rate decreased from the aerobic value of 5.3 to less than 2 nmol/10(6) cells per min in anoxic cells; half-maximal change occurred at 5 microM O2. Although cells could not maintain normal thiol and NADPH pools at infusion rates above the critical value, analysis of the rates of thiol depletion showed that the maximal NADPH supply rate for GSSG reduction under aerobic conditions was 7-8 nmol/10(6) cells per min and was affected by hypoxia to the same degree as the critical value. Thus, hypoxia and anoxia impair the capability of cells to supply NADPH for the reduction of thiol oxidants. This could be an important factor in the sensitivity of hypoxic and ischemic tissues to oxidative injury.