Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells. Sequestration of Na(+) ions into the vacuole is one mechanism to confer Na(+)-tolerance on these organisms. In the present study we provide direct evidence that the Arabidopsis thaliana At-NHX1 gene and the yeast NHX1 gene encode low-affinity electroneutral Na(+)/H(+) exchangers. We took advantage of the ability of heterologously expressed At-NHX1 to functionally complement the yeast nhx1-null mutant. Experiments on vacuolar vesicles isolated from yeast expressing At-NHX1 or NHX1 provided direct evidence for pH-gradient-energized Na(+) accumulation into the vacuole. A major difference between NHX1 and At-NHX1 is the presence of a cleavable N-terminal signal peptide (SP) in the former gene. Fusion of the SP to At-NHX1 resulted in an increase in the magnitude of Na(+)/H(+) exchange, indicating a role for the SP in protein targeting or regulation. Another distinguishing feature between the plant and yeast antiporters is their sensitivity to the diuretic compound amiloride. Whereas At-NHX1 was completely inhibited by amiloride, NHX1 activity was reduced by only 20-40%. These results show that yeast as a heterologous expression system provides a convenient model to analyse structural and regulatory features of plant Na(+)/H(+) antiporters.