The effectiveness of alkylating agents in the treatment of ovarian cancer is limited by the frequent development of drug resistance. In order to examine the mechanisms of resistance and potential ways in which this resistance could be overcome, we have developed a human ovarian cancer cell line, 1847ME, resistant to the bifunctional amino acid nitrogen mustard, melphalan. A 4-fold higher concentration of melphalan was required to produce an equivalent reduction in tumor colony formation in 1847ME cells as compared to the parent melphalan-sensitive line A1847. The magnitude of resistance in 1847ME was similar to that observed in the cell lines NIH:OVCAR-2, NIH:OVCAR-3, and NIH:OVCAR-4 which were derived from ovarian cancer patients clinically resistant to alkylating agents. There was no detectable difference in melphalan uptake between A1847 and 1847ME. The cellular content of the inactive dihydroxy melphalan metabolite, however, was two times greater in 1847ME compared to A1847. Levels of the principal intracellular thiol, glutathione, were found to be 2-fold greater in 1847ME than in A1847, and to be similarly elevated in the OVCAR lines. Depletion of glutathione by incubation of the cells in cystine-free medium or in the presence of the specific inhibitor of glutathione synthesis, DL-buthionine-S,R-sulfoximine, was accompanied by a marked increase in melphalan cytotoxicity. Doses of DL-buthionine-S,R-sulfoximine which were only minimally cytotoxic were associated with complete reversal of the induced resistance to melphalan in 1847ME. Synergism between melphalan and DL-buthionine-S,R-sulfoximine was also demonstrated in the OVCAR cell lines derived from previously treated ovarian cancer patients. The reversal of induced resistance to melphalan by modulation of glutathione levels is of potential clinical relevance. In addition, these cell lines provide a useful model system in which to study further the mechanisms of alkylating agent resistance in human tumors.