Acquired drug resistance is a major obstacle in the successful treatment of cancer by chemotherapy. To study mechanisms of resistance to alkylating agents, we have derived an in vitro subline of the murine KHT-iv sarcoma, KHT-rcp/iv, that has acquired resistance to cyclophosphamide. Compared to the parental line, KHT-rcp/iv exhibited an approximately 2.8-fold resistance, determined from the ratio of the slopes of the resistant to the parental survival curve. The subline also was found to be cross-resistant to other alkylating agents, as well as to the epipodophyllotoxin, etoposide, a structurally unrelated compound (resistance factor of approximately 2.2). In view of this broad spectrum of cross-resistance observed, we investigated the possibility that glutathione (GSH), an abundant tripeptide implicated in the detoxification of electrophilic species, may be involved. GSH levels were increased two-fold in the resistant line (16.0 +/- 1.0 fmol/cell) as compared to the parent line (8.1 +/- 0.7 fmol/cell). Also, when the GSH content was reduced to that of the parent with the addition of the thiol depletor buthionine sulfoxinine (BSO), the sensitivity of the subline to cyclophosphamide, as well as to the other agents, was enhanced so that it equaled that of the parent cell line. These results imply that enhanced GSH levels may be a major mechanism of resistance in the KHT-rcp/iv cells. Our findings further suggest that thiol manipulation may prove useful in overcoming this type of pleiotropic drug resistance.