Yeast complementation experiments were carried out to define the possible function of PeNhaD1, a Na(+)/H(+) antiporter gene from Populus euphratica Oliv., a salt resistant tree. PeNhaD1 was introduced to the Saccharomyces cerevisiae mutant strain ANT3 (Deltaena1-4::HIS3 Deltanha1::LEU2), which lacks the plasma membrane Na(+)/H(+) antiporter gene ScNHA1 or GX1 (Deltanhx1::TRP1), which lacks tonoplast Na(+)/H(+) antiporter gene ScNHX1. Our results showed that PeNhaD1 rescued the normal growth of ANT3 in the presence of high salt (80 mmol/L NaCl on solid medium or 400 mmol/L in liquid medium, pH 6.0), but not that of GX1, suggesting that PeNhaD1 may play a role in salt tolerance of Populus euphratica by maintaining the capacity for salt exclusion under saline condition.