Although melphalan has been used as a therapeutic reagent for multiple myeloma, many patients become refractory. To elucidate the mechanism of resistance to melphalan, we generated a melphalan-resistant myeloma cell line, KHM-11(EMS), by treating a parental line, KHM-11, with a mutagen, ethylmethanesulfonate. KHM-11(EMS) is 55 times more resistant to melphalan. gamma-Glutamylcysteine synthetase, P-glycoprotein, multidrug-resistance-associated protein, lung-resistance-related protein and the Bcl-2 family of proteins were not responsible for the drug resistance in KHM-11(EMS). Intracellular incorporation of melphalan to myeloma cells was determined by using [(14)C]-labeled melphalan. Accumulation of melphalan in KHM-11(EMS) was 43% of KHM-11, while the efflux rates were comparable in both cell lines. The uptake of melphalan was inhibited by the addition of L-phenylalanine, indicating that melphalan is incorporated through the L-phenylalanine transporter as reported previously. Expression of CD98, which was recently cloned as an L-phenylalanine transporter, was 6-fold decreased in KHM-11(EMS), suggesting that CD98 may be correlated with the incorporation of melphalan. CD98 expression and incorporation of melphalan were analyzed in fresh purified myeloma cells from 5 patients. All myeloma cells from 4 cases expressed CD98 at a high level and incorporated melphalan. However, tumor cells from 1 case expressed CD98 at low levels and did not incorporate melphalan. Taken together, reduced melphalan uptake could be responsible for the drug resistance in KHM-11(EMS), and down-regulation of CD98 may be related to this phenomenon. Further investigation of the correlation between impaired drug uptake and down-regulation of CD98 in myeloma cells should be important to understand the mechanism of resistance to melphalan.