Although it has been shown previously that the depletion of cellular thiols increases doxorubicin cytotoxicity, the mechanism of sensitization is not clear. To study this question, the effect of D,L-buthionine-S,R-sulfoximine (BSO) on doxorubicin cytotoxicity and the stabilization of DNA-topoisomerase II complexes (cleavable complexes) was investigated in V79 cells. Incubations with BSO (10 mM) were for 5 hr beginning 4 hr prior to doxorubicin exposure since a 4 hr incubation with 10 mM BSO is known to decrease glutathione levels below 5% of control V79 cells. These BSO pre-treatments increased doxorubicin cytotoxicity. At doxorubicin concentrations of 5 μg/ml, BSO resulted in an 8-10 fold decrease in surviving cells, compared to cells exposed to doxorubicin alone. It was determined that BSO pre-treatments did not affect the accumulation of doxorubicin into the cell, the rate of cleavable complex stabilization by doxorubicin, or the rate of dissociation of stabilized cleavable complexes. These data suggest that BSO-induced doxorubicin sensitization occurs at a step following the stabilization of cleavable complexes or by an independent mechanism. © 1992.