The production of semiquinone free radicals has been measured by electron paramagnetic resonance spectroscopy (EPR) in Chinese hamster ovary cells in which 7-hydroxy daunorubicin aglycone had been incorporated. The highly lipophilic daunorubicin aglycone was incorporated into the cellular membrane by swirling a cell suspension over a thin layer of daunorubicin aglycone. Thus, the observed semiquinone free radical was likely formed directly in the lipophilic environment of the cellular membrane. The linewidth of the observed EPR signal suggested that a neutral protonated semiquinone species was formed. In the presence of the cell-impermeant paramagnetic line broadening agent chromium(III) oxalate, no detectable signal was observed. This result indicates that even though the semiquinone is embedded in the membrane, it is still partly accessible to the external chromium(III) oxalate. Analysis of chloroform extracts of the cells after EPR experiments indicated that daunorubicin aglycone was extensively metabolized. The results of a growth inhibition assay carried out on cells into which daunorubicin aglycone had been incorporated showed almost no effect on cell growth. This result indicates that in spite of significant daunorubicin aglycone-induced radical formation taking place directly in the cell membrane, little cell damage results.