Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na(+),K(+)-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine beta1 subunit become adhesive, and those homotypic interactions amongst beta1 subunits of the Na(+),K(+)-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the alpha subunit of the Na(+),K(+)-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the alpha subunit. In this study, we investigated whether the adhesion between beta1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the beta1 subunit of the Na(+),K(+)-ATPase (CHO beta1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO beta1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the beta1 subunit of the Na(+),K(+)-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO beta1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO beta1 cells. Collectively, our findings suggest that the beta1 subunit adhesion is modulated by the expression levels of the Na(+),K(+)-ATPase at the plasma membrane, which is regulated by ouabain.