In plants, the plasma membrane Na+/H+ antiporter is the only key enzyme that extrudes cytosolic Na+ and contributes to salt tolerance. But in fungi, the plasma membrane Na+/H+ antiporter and Na+-ATPase are known to be key enzymes for salt tolerance. Saccharomyces cerevisiae Ena1p ATPase encoded by the ENA1/PMR2A gene is primarily responsible for Na+ and Li+ efflux across the plasma membrane during salt stress and for K+ efflux at high pH and high K+. To test if the yeast ATPase would improve salt tolerance in plants, we expressed a triple hemagglutinin (HA)-tagged Ena1p (Ena1p-3HA) in cultured tobacco (Nicotiana tabacum L.) cv Bright Yellow 2 (BY2) cells. The Ena1p-3HA proteins were correctly localized to the plasma membrane of transgenic BY2 cells and conferred increased NaCl and LiCl tolerance to the cells. Under moderate salt stress conditions, the Ena1p-3HA-expressing BY2 clones accumulated lower levels of Na+ and Li+ than nonexpressing BY2 clones. Moreover, the Ena1p-3HA expressing BY2 clones accumulated lower levels of K+ than nonexpressing cells under no-stress conditions. These results suggest that the yeast Ena1p can also function as an alkali-cation (Na+, Li+, and K+) ATPase and alter alkali-cation homeostasis in plant cells. We conclude that, even with K+-ATPase activity, Na+-ATPase activity of the yeast Ena1p confers increased salt tolerance to plant cells during salt stress. (C) 2004 Wiley Periodicals, Inc.