Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.