A new YNB medium containing very low concentrations of alkali metal cations has been developed to carry out experiments to study potassium homoeostasis. Physiological characterization of Saccharomyces cerevisiae BY4741 strain and the corresponding mutant lacking the main potassium uptake systems (trk1 trk2) under potassium nonlimiting and limiting concentrations was performed, and novel important differences between both strains were found. At nonlimiting concentrations of KCl, the two strains had a comparable cell size and potassium content. Nevertheless, mutants were hyperpolarized, had lower pH and extruded fewer protons compared with the BY4741 strain. Upon transfer to K(+)-limiting conditions, cells of both strains became hyperpolarized and their cell volume and K(+) content diminished; however, the decrease was more relevant in BY4741. In low potassium, trk1 trk2 cells were not able to accomplish the cell cycle to the same extent as in BY4741. Moreover, K(+) limitation triggered a high-affinity K(+)/Rb(+) uptake process only in BY4741, with the highest affinity being reached as soon as 30 min after transfer to potassium-limiting conditions. By establishing basic cellular parameters under standard growth conditions, this work aims to establish a basis for the investigation of potassium homoeostasis at the system level.