Oleandrin, a cardiac glycoside component of Nerium oleander, has been shown to induce apoptosis in malignant cells. While human tumor cells are very sensitive to growth inhibition by oleandrin, murine tumor cells are extremely resistant. Using human BRO and mouse B16 melanoma cell lines, we explored several possible determinants of cell sensitivity to oleandrin and compared with ouabain. The studies include Na+, K(+)-ATPase activity and its isoforms as well as the cellular uptake of these cardiac glycosides. Oleandrin and ouabain induced apoptosis was detected in BRO cells while no evidence of cell death was observed in B16 cells even at concentrations 1000-fold higher than that used for BRO cells. Cellular uptake of oleandrin and ouabain was 3-4 fold greater in human BRO tumor cells than murine tumor cells. Partially purified Na+, K(+)-ATPase from human BRO cells was inhibited at a concentration that was 1000-fold less than that was required to inhibit mouse B16 enzyme to the same extent. Using Western blot analyses, human BRO cells were found to express both the sensitive alpha3 isoform and the less sensitive alpha1 isoform of Na+, K(+)-ATPase while mouse B16 cells expressed only the alpha1 isoform. These data suggest that differential expressions of Na+, K(+)-ATPase activities and its isoforms in BRO and B16 cells as well as cellular drug uptake may be important determinants of tumor cell sensitivity to cardiac glycosides.