The effect of cycloheximide (an inhibitor of cellular protein synthesis) on doxorubicin-induced cytotoxicity in V79 (rodent fibroblasts) cells was investigated. Cycloheximide is a potent protector of doxorubicin-induced cytotoxicity at concentrations paralleling those required for protein synthesis inhibition. The greatest protective effect was achieved at 10 microM cycloheximide; this concentration correlated with 95% inhibition of protein synthesis. A 15 minute cycloheximide (10 microM) exposure resulted in maximal protein synthesis inhibition; however, 4-6 hr of pretreatment with cycloheximide (10 microM) was required to maximally protect cells from doxorubicin. These results suggest that a time-dependent depletion of a protein is required for cycloheximide's protective effect. Cycloheximide treatments were found to decrease intracellular accumulation of doxorubicin by 35-50% but this decrease accounts for only a small fraction of the total protective effect. When corrections were made for differences in doxorubicin accumulation, cycloheximide had no effect on the formation of DNA-protein crosslinks (DNA-topoisomerase II complexes revealed as single strand DNA breaks in alkaline elution studies). These studies suggest that cycloheximide confers protection from doxorubicin cytotoxicity by a step which occurs following the stabilization of DNA-topoisomerase II complexes.