Human ovarian tumors from A2780 cells were grown as xenografts in immunodeficient mice, treated with a single i.p. dose of melphalan and tumor cells were removed and placed into tissue culture. The cells from the treated tumors exhibited an approximately 2-fold resistance to melphalan in vitro compared to cells taken from untreated tumors. This degree of resistance was similar to that of cells from tumors formed from melphalan-resistant A2780-ME cells. The cells from the treated tumors were also resistant to cisplatin but not to doxorubicin. They contained approximately 2-fold higher levels of glutathione than cells from the untreated tumors. Exposure of the cells to buthionine sulfoximine (a specific inhibitor of glutathione biosynthesis) eliminated the difference in glutathione levels as well as the difference in sensitivity to melphalan. When tumor-bearing animals were treated with buthionine sulfoximine in addition to melphalan the resulting tumor cells were not resistant to the drug. Resistance could also be demonstrated in the tumors themselves in vivo: the growth of previously untreated tumors was severely inhibited by a high dose of melphalan (11.7 mg/kg) administered i.p. to the animals, whereas the growth of tumors which had received prior treatment with melphalan was unaffected by the subsequent high dose. The rapid development of drug-resistant tumor cells after a single drug treatment in vivo makes this an excellent system for the investigation of the mechanisms by which resistance develops as well as for use in the screening for agents which can prevent it.