The plasma membrane Na⁺, K⁺-ATPase which drives potassium into and sodium out of the cell has important roles in numerous physiological processes. Cardiac steroids, (CS) such as ouabain and bufalin, specifically interact with the pump and affect ionic homeostasis, signal transduction and endocytosed membrane traffic. CS-like compounds are present in mammalian tissues, synthesized in the adrenal gland, and considered to be new family of steroid hormones. In this study the mechanism of Na⁺, K⁺-ATPase involvement in the regulation of endocytosis, is explored. We show that the effects of various CS on changes in endosomal pH are mediated by the pump and correspond to their effects on endosomal membrane traffic. In addition, it was found that CS-induced changes in endocytosed membrane traffic were dependent on alterations in [Na⁺] and [H⁺] in the endosome. Furthermore, it is shown that various CS differentially regulate endosomal pH and membrane traffic. The results suggest that these differences are due to specific binding characteristics. Based on our observations we propose that Na⁺, K⁺-ATPase is a key player in the regulation of endosomal pH and endocytosed membrane traffic. Furthermore, our results raise the possibility that CS-like hormones regulate differentially intracellular membrane traffic.