In this study, we have examined the relationship between sensitivity to mitomycin C (MMC) and glutathione (GSH) and glutathione transferase (GST) levels using a panel of three unrelated human bladder cancer cell lines. J82, HT-1197, and SCaBER. Cell lines HT-1197 and SCaBER were about 2- and 4.5-fold more resistant to MMC as compared to J82. Although the GSH level did not differ significantly in these cell lines, GST activity in HT-1197 and SCaBER cells were higher by about 2.3- and 6.0-fold, respectively, as compared to J82. Similar to GST activity, GST pi content was highest in the most insensitive cell line and lowest in J82 cells. The cytotoxicity of MMC was increased significantly in these cells by a 1-h pretreatment with a nontoxic concentration of ethacrynic acid (EA), an inhibitor of GST activity. EA pretreatment resulted in a marked GSH depletion as well as GST activity inhibition in both of these cells. Although pretreatment of J82 and SCaBER cells with a nontoxic concentration of D,L-buthionine-S,R-sulfoximine (BSO) caused similar GSH depletion, the cytotoxicity of MMC was enhanced only in SCaBER cells. The differential effect of BSO on MMC cytotoxicity in these cell lines appeared to be due to the differences in the extent of GSH regeneration after removal of BSO. While a marked GSH regeneration occurred in J82 cells within 1 h after BSO removal, such an effect was not observed in SCaBER cells. Combined treatment of these cells with BSO and EA produced a greater potentiation of MMC cytotoxicity in both the cell lines when compared to BSO or EA treatment alone. We conclude that GSH/GST levels may affect the sensitivity of human bladder cancer cells to MMC.