There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.