In cereals, a common salinity tolerance mechanism is to limit accumulation of Na(+) in the shoot. In a cross between the barley variety Barque-73 (Hordeum vulgare ssp. vulgare) and the accession CPI-71284 of wild barley (H. vulgare ssp. spontaneum), the HvNax3 locus on chromosome 7H was found to determine a ~10-25 % difference in leaf Na(+) accumulation in seedlings grown in saline hydroponics, with the beneficial exclusion trait originating from the wild parent. The Na(+) exclusion allele was also associated with a 13-21 % increase in shoot fresh weight. The HvNax3 locus was delimited to a 0.4 cM genetic interval, where it cosegregated with the HVP10 gene for vacuolar H(+)-pyrophosphatase (V-PPase). Sequencing revealed that the mapping parents encoded identical HVP10 proteins, but salinity-induced mRNA expression of HVP10 was higher in CPI-71284 than in Barque-73, in both roots and shoots. By contrast, the expression of several other genes predicted by comparative mapping to be located in the HvNax3 interval was similar in the two parent lines. Previous work demonstrated roles for V-PPase in ion transport and salinity tolerance. We therefore considered transcription levels of HVP10 to be a possible basis for variation in shoot Na(+) accumulation and biomass production controlled by the HvNax3 locus under saline conditions. Potential mechanisms linking HVP10 expression patterns to the observed phenotypes are discussed.