The cellular actions of ouabain are complex and involve different pathways, depending on the cell type and experimental conditions. Several studies have reported that Madin-Darby canine kidney (MDCK) cellular sensitivity to ouabain is not related to Na-K-ATPase inhibition, and others showed that some cell types, such as Ma104, are resistant to ouabain toxicity albeit their Na-K-ATPase isoforms possess high affinity for this glycoside. We describe here that the effects of ouabain and ouabagenin also diverge in MDCK and Ma104 cells, being MDCK cells more resistant to ouabagenin, while Ma104 cells are resistant to both molecules. This feature seems to correlate with induction of cell signaling, since ouabain, but not ouabagenin, induced an intense and sustained increase in tyrosine phosphorylation levels in MDCK cells. Moreover, ouabain-induced phosphorylation in Ma104 cells was approximately half than that observed in MDCK cells. The proportion between alpha and beta subunits of Na-K-ATPase was similar in MDCK cells, though Ma104 cells presented more alpha subunits, located mainly at the cytoplasm. Furthermore, a fluorescent ouabain-analog labeled mainly the cytoplasm of Ma104 cells, the opposite of that seen in MDCK cells, corroborating the results using anti-Na-K-ATPase antibodies. Hence, the results suggest that ouabain and ouabagenin differ in terms of Na-K-ATPase inhibition and cell signaling activation in MDCK cells. Additionally, MDCK and Ma104 cell lines respond differently to ouabain, perhaps due to an intrinsic ability of this glycoside to selectively reach the cytoplasm of Ma104 cells.