SummaryThe Arabidopsis thaliana vacuolar Na+/H+ antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na+/H+ exchangers led to the identification of five additional AtNHX genes (AtNHX2–6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower‐abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT–PCR. AtNHX1, 2 or 5 suppress, with differential efficacy, the Na+/Li+‐sensitive phenotype of a yeast mutant that is deficient in the endosomal/vacuolar Na+/H+ antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na+ ion compartmentalization and maintain intracellular K+ status. Seedling steady‐state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi‐osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper‐osmotic up‐regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady‐state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2‐1, mutant indicating that the osmotic responsiveness of these genes is ABA‐dependent. An as yet undefined stress signal pathway that is ABA‐ and SOS‐independent apparently controls transcriptional up‐regulation of AtNHX5 expression by hyper‐saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na+.