Cells depleted of their glutathione (GSH) by treatment with L-buthionine sulfoximine (BSO) are more sensitive to ionizing radiation and chemotherapeutic agents. To assess the effects of GSH depletion on repair of radiation-induced DNA damage, we have determined DNA-protein cross-links (DPC) in A549 cells by a nitrocellulose filter binding assay. Untreated A549 cells have a low level of DPC (0.7%), whereas cells incubated with BSO for 92 hours have an elevated level of DPC (1.5%). The dose response for production of radiation-induced DPC is approximately 0.4% DNA bound per 10 Gy for both BSO-treated and non-pretreated cells. Cells not exposed to BSO repair 85% of the radiation-induced DPC in 4 hours. Less repair (55%) is observed for BSO-treated cells incubated for 4 hours postirradiation in conditioned medium, and DPC repair is nearly completely blocked if GSH-depleted cells are given fresh medium during the 4-hour repair interval. The DPC repair is not influenced by the pH of the media between 6.6 and 7.4. Data indicate that intracellular GSH regulates the level of a sulfhydryl-containing repair enzyme, and that other, as yet undefined, nutrients may further influence the steady-state level of DPC.