The kidney of vertebrates plays a major role in the homeostasis of the extracellular fluid. Despite large changes in salt intake, the kidney is able to maintain the extracellular osmolarity and volume within very narrow margins. During the evolution of vertebrates, aldosterone played a critical physiological role about 300 million years ago with the emergence of amphibia, the first vertebrates to adapt to a terrestrial environment. Comparative studies of physiology, biochemistry, and molecular biology have helped in delineating the most significant steps involved. In the present study we have focused on the evolution of ENaC and Na,K-ATPase, which are the key effectors and limiting factors of the aldosterone response in the mammalian distal nephron. By searching for homologs in various eukaryotes, from unicellular eukaryotes (“protists”) to multicellular metazoan, we provide here a novel view of how mammalian aldosterone-dependent control of sodium homeostasis, blood volume and blood pressure might have evolved. We propose that Na,K-ATPase emergence, together with ENaC/Degenerin, is linked to the development of multicellularity in the Metazoan kingdom. The establishment of multicellularity and the associated extracellular compartment (“internal milieu”) precedes the emergence of other key elements of the aldosterone signaling pathway.