The cardiac glycoside ouabain, which is a specific inhibitor of the Na+, K+-pump, confers dramatic protection from the cytotoxic effects of doxorubicin (Adriamycin). This effect was documented in cultured A549 cells (human lung adenocarcinoma). CCL210 cells (human fibroblasts), and V79 cells (hamster fibroblasts). Maximum protection from doxorubicin cytotoxicity was achieved using 1 µm ouabain for A549 and CCL210 cells and 300 µm ouabain for V79 cells. These concentrations correlated well with the concentrations of ouabain required to induce Na+, K+-pump blockade, which was assessed using the K+ analogue 86Rb+. This suggests that protection is mediated by pump blockade. Addition of ouabain at the same time as doxorubicin was just as protective as preincubation with ouabain for an hour, demonstrating that the ouabain acts rapidly. Ouabain treatment affected neither influx nor efflux of doxorubicin. Ouabain also had no effect on verapamil-induced inhibition of doxorubicin efflux. However, ouabain partially blocked the verapamil-induced potentiation of the cytotoxic effects of doxorubicin. Therefore, ouabain does not protect by affecting intracellular doxorubicin levels. Fluorescence microscopy showed that the ability of doxorubicin to reach the nucleus was not influenced by ouabain. Alkaline elution studies demonstrated that ouabain greatly decreased doxorubicin-induced DNA strand breakage. Protection from cytotoxicity correlated closely with this decrease in strand breakage. These studies suggest that the stabilization of DNA-topoisomerase II complexes is closely linked to the mechanism of doxorubicin cytotoxicity and that this stabilization is influenced by the intracellular ionic milieu.