The carcinogen 4-nitroquinoline 1-oxide (4-NQO) was found to rapidly deplete non-protein thiols (NPSH) from Ehrlich ascites tumor cells and V79 Chinese hamster fibroblasts. The effects of NPSH on 4-NQO metabolism were studied by measuring 4-hydroxyaminoquinoline 1-oxide formation, CN- -insensitive oxygen consumption, and reduction of ferricytochromes c + c1 in normal cells and in cells pretreated with the thiol reagent N-ethylmaleimide. Removal of thiols before treatment with 4-NQO resulted in increased production of 4-hydroxyaminoquinoline 1-oxide and increased production of nitro radicals. The NPSH thus appeared to play a significant role in 4-NQO detoxification. Glutathione, when present in culture medium during 4-NQO treatment, protected V79 cells from 4-NQO toxicity. Several mechanisms for reaction of 4-NQO with intracellular NPSH were indicated. Both V79 and Ehrlich cells contained appreciable amounts of glutathione S-transferase (EC 2.5.1.18), which catalyzes the nucleophilic substitution of the nitro group of 4-NQO with thiols. Greater thiol loss under oxic than under hypoxic conditions suggested oxidation by superoxide, peroxide, or hydroxyl radical formed in the course of 4-NQO reduction. In addition, reaction of thiols with nitro radicals or with nitrosoquinoline 1-oxide was indicated by the inhibitory effect of glutathione on oxygen consumption in solutions of 4-NQO and sodium ascorbate.