Glutathione (GSH) and glutathione-S-transferases (GST) have been implicated in resistance of tumor cells to certain alkylating agents, including melphalan. Glutathione levels and GST activities were determined in melphalan-resistant sublines of the human prostate carcinoma cell lines DU 145, PC-3 and LNCaP produced by serial treatment with melphalan at progressively increasing concentrations. The resistant sublines M4.5DU145, M5DU145, M6DU145, M6PC-3 and M6LNCaP were 27-, 7-, 3-, 6- and 2-fold more resistant to melphalan than the parental lines. The melphalan-resistant DU 145 and PC-3 lines showed cross-resistance to cisplatin and tetraplatin, but retained sensitivity to vinblastine, colchicine and etoposide. Interestingly, both sublines were also resistant to methotrexate and adriamycin. The melphalan-resistant LNCaP line showed slight resistance to cisplatin and adriamycin, but remained sensitive to tetraplatin and methotrexate. This line also retained sensitivity to vinblastine while developing resistance to colchicine. Intracellular GSH levels were increased 2.8 fold for M5DU145, 1.7 fold for M6PC-3 and 2.1 fold for M6LNCaP compared to the parental lines, whereas GST activity using chlorodinitro-benzene as a substrate was comparable for all lines. When cumene hydroperoxide was used as a substrate, an increase in GST activity was noted only in the M6PC-3 line as compared with the parent line. Western blot analysis showed no change in GST isozyme profile between parent and resistant DU 145 lines; however a mu class isoenzyme was detected in the resistant, but not in the parent PC-3 line, using a Yb1 antibody. M5DU145 cells maintained in the absence of melphalan for seven months maintained their resistance to melphalan. Depletion of GSH, with buthionine sulfoximine, to control levels reversed melphalan resistance to control levels.