We studied the effects of ifosfamide and major metabolites on intracellular glutathione (GSH) levels in human peripheral blood lymphocytes (PBL). In vitro exposure of PBL to 4-hydroperoxyifosfamide (4-OOH-IF), acrolein or chloroacetaldehyde at 37 degrees C for 60 min led to a concentration dependent depletion of intracellular GSH. The concentration of the three metabolites to cause a 50% depletion of GSH in PBL was in the micromolar range (acrolein: 16 +/- 4 microM; 4-OOH-IF: 22 +/- 9 microM; chloroacetaldehyde: 30 +/- 7 microM). Exposure to ifosfamide, the non-activated drug, had no effects on the intracellular GSH levels. Pretreatment with 4-OOH-IF suppressed dose-dependently the interleukin-2-induced proliferation of PBL. Incubation of PBL together with 2-mercaptoethanesulfonate (mesna) and 4-OOH-IF, acrolein or chloroacetaldehyde prevented the GSH depletion. The protecting effect of mesna in combination with 4-OOH-IF was independent of GSH biosynthesis, because addition of buthionine sulfoximine had no significant influence on this effect. These findings indicate a novel protective mechanism of mesna against intracellular GSH depletion of PBL during exposure to metabolites of ifosfamide.