We present data describing a human myeloma cell line (8226/LR-5) selected for resistance to melphalan which exhibits a 7-fold level of resistance to melphalan and is partially cross-resistant to other bifunctional alkylators and X-irradiation. Melphalan resistance is relatively unstable with a decrease in resistance observed within 17 weeks in the absence of drug. The resistance observed in this cell line is not mediated by reduced intracellular melphalan accumulation. DNA interstrand cross-linking at equivalent intracellular drug accumulation is significantly reduced in the resistant subline. Whether this reduction is the result of a decrease in the formation of this lesion or to an increased rate of removal of the lesion remains to be determined. Growth characteristics and cell cycle kinetics, including S phase, were similar between sensitive and resistant cell lines. Intracellular nonprotein thiols were found to be significantly elevated in the resistant 8226/LR-5 cells; as cells revert or lose resistance, intracellular nonprotein sulfhydryl levels decline. Prior treatment of the cells with buthionine sulfoximine significantly reduced nonprotein sulfhydryl levels and enhanced melphalan cytotoxicity in both the sensitive and resistant cell lines. Thiols appear to play a role in mediating melphalan resistance.