HL-60-R, a multi-drug-resistant (MDR) subclone of the human leukemia cell line HL-60, was selected in continuous culture in doxorubicin (DOX) in the absence of mutagenic agents. When compared to the parent line HL-60, HL-60-R showed greater relative resistance to vinblastine than to etoposide, or to the selecting agent DOX. Co-exposure to verapamil, a known modulator of MDR, partially increased its sensitivity to DOX and vinblastine. The HL-60-R cell line stained positively with the P-glycoprotein-specific monoclonal antibody (MAb), C219, whereas the HL-60 parent was negative. Southern analysis showed 32-fold amplification of the mdrI gene in HL-60-R, whereas slot-blot analysis demonstrated 70-fold over-expression of the specific mdrI message in HL-60-R compared to HL-60. Northern blot analysis revealed the presence of 2 species of messenger RNA of sizes 5.1 kb and 4.5 kb. No transcripts were detectable in the parent. Flow cytometric analysis showed significantly reduced cellular retention of DOX as well as rapid efflux from the drug-resistant cell line. HL-60-R proved to be nearly 4 times more resistant to hydrogen peroxide than its parent, and 1,000 times more resistant to inhibition of cellular glutathione synthesis by D,L-buthionine sulfoximine (BSO). Verapamil modulated DOX resistance in HL-60-R incompletely but, in the presence of glutathione depletion, nearly completely reversed DOX resistance. Elevated levels of glutathione and glutathione-peroxidase activity were demonstrated, thereby implicating enhanced activity of the glutathione/glutathione-peroxidase cycle as an additional basis for its resistance to DOX. These findings suggest that an enhanced capacity for detoxifying oxyradicals may contribute to anthracycline resistance in acute leukemia.