The ability of astroglial cells to detoxify exogenously applied tertiary butyl hydroperoxide (tBHP) was tested using astroglia-rich primary cultures derived from the brains of newborn rats. If 200 microM tBHP was applied, this compound disappeared from the incubation buffer with an apparent half-life of about 5 min. After 20 min incubation tBHP was not detectable any more. A decay of tBHP was found even in the absence of cells. Therefore, half-times for the cell-dependent tBHP clearance were corrected for the cell-independent decay of tBHP. The cell-dependent half-time of tBHP in the incubation buffer was found strongly elevated i) with increasing concentration of tBHP, ii) after decrease of the glutathione content of the cells by a preincubation with buthionine sulfoximine, an inhibitor of glutathione synthesis, iii) in the presence of mercaptosuccinate, an inhibitor of glutathione peroxidase, and iv) in the absence of glucose, the precursor for the generation of NADPH. Incubation of astroglial cells with 200 microM tBHP in the absence of glucose led to a 46% oxidation of the cellular glutathione within 30 s. Under these conditions the cells were unable to restore the original high ratio of the concentrations of GSH to GSSG within 30 min of incubation. In contrast, if glucose was present the level of GSSG encountered on incubation with tBHP was lower (32% of total glutathione after 30 s) and the original ratio of the levels of GSH to GSSG was essentially reestablished within 10 min. In the presence of 3 mM mercaptosuccinate oxidation of glutathione was almost completely inhibited. These results demonstrate that an exogenous hydroperoxide is detoxified rapidly by astroglial cells via the glutathione system.