Cellular oxidative stress is associated with such pathological conditions as arteriosclerosis, inflammatory diseases and cancer. The oxidation of the biomarkers. 2',7'-dichlorofluorescin (DCFH), 2-deoxyribose, and lipid peroxidation are often used to assess the status of oxidative stress in cells and tissues. Since high levels of reduced glutathione (GSH) and acidic conditions have been associated with diminished chemical lethality, we evaluated the influence of these parameters on the cellular response to oxidative stress. We used a cultured hepatocyte line (ch/ch cells) that is susceptible to oxidative toxicity. A hydroxyl radical-generating system consisting of H2O2, ascorbate and iron produced a pH-dependent lethality, with complete cell killing at pH 7.4 and none at pH 6.8. Lethality correlated with the depletion of intracellular GSH, and with an increase in DNA fragmentation. The influence of GSH and pH was assessed for DCFH and 2-deoxyribose oxidation, and for lipid peroxidation. The oxidation of DCFH and 2-deoxyribose was inhibited by GSH, with about 4-fold greater inhibition efficacy at pH 6.8 than at pH 7.4 [IC50 values (microM GSH) for pH 6.8 and 7.4, respectively: DCFH = 7 and 30; 2-deoxyribose = 125 and 490]. GSH did not affect lipid peroxidation at either pH, even at a high intracellular concentration of 10 mM. We conclude: 1) GSH is not inhibiting DCFH and 2-deoxyribose oxidation by simply quenching reactive oxygen (hydroxyl radical or perferryl oxygen), since GSH did not inhibit lipid peroxidation: 2) the protonated form GSH is more likely to be the inhibitory species rather than GS-, since even in the simple cell-free systems lower pH inhibited biomarker oxidation; and; 3) hydroxyl radical may not be the primary intracellular oxidant of DCFH, since intracellular GSH concentrations are typically 10- to 100-fold higher than the IC50 values for GSH inhibiting reactive oxygen-mediated DCFH oxidation.