It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and -extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [3H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [3H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (51Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seen to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.