A model system for the detection of reactive metabolites, using glutathione depletion after microsomal activation, has been described previously. We developed a battery of complementary test systems using rat liver microsomes for metabolism and aqueous glutathione solutions, human erythrocytes or hemolysate derived therefrom, as target. Reactive metabolite formation and the ability of metabolites to pass the erythrocyte membrane were tested using 3-hydroxyacetanilide (3-HAA) and cyclophosphamide (CP) as substrates. Neither unchanged 3-HAA nor CP depleted glutathione in erythrocytes or in aqueous reduced glutathione solutions (GSH solutions). Addition of fortified normal or liver microsomes from rats pretreated with phenobarbital (PB microsomes) induced a 3-HAA/CP concentration-dependent glutathione depletion in both systems. With PB microsomes, higher depletions were found. While unchanged 3-HAA did not deplete aqueous GSH solutions or glutathione in erythrocytes, a significant depletion in hemolysate was found. The results indicate that both CP and 3-HAA metabolites are able to pass through the erythrocyte membrane. While both substances can metabolically be activated by rat liver microsomes, only 3-HAA can be activated by soluble factors in erythrocytes. However, unchanged 3-HAA has no effect on GSH in erythrocytes. This might be caused by an inability of unchanged 3-HAA to enter the erythrocyte. More generally, an adequate combination of the test systems described can be used to detect (a) the reactivity of unchanged substances and their metabolites, and (b) the ability of unchanged substances and their reactive metabolites to pass through the erythrocyte membrane.