Yeast Nhx1p is a vacuolar N+(K+)/H+ antiporter controlling cellular pH and vesicle trafficking.Our study indicates that Nhx1p plays an important role in yeast vacuole fusion.We find that the nhx1p △ cells show vacuole fragmentation phenotypes and have reduced vacuole fusion activities (35% reduction relative to wild type (MMT)), indicating that Nhx1p is involved in vacuole fusion in yeast.Further study shows that the WT Nhx1p proteins could complement the mutant phenotype of the nhx1p△ cells and recover the fusion activity to the WT levels; however, complement with a mutant protein (D201N) defective in ion transport activity and vesicle trafficking does not rescue the mutant phenctype and fusion activity, suggesting that the ion transport activity of the Nhx1p is important for the yeast vacuole fusion.Treating vacuoles with weak base, chloroquine rescues mutant fusion activity to WT levels, further demonstrating that Nhx1p regulates yeast vacuole fusion through controlling the luminal pH.Vacuole fusion activity in the nhx1p△ cells could be rescued by the soluble SNARE Vam7p, but Vam7p only stimulates the first round fusion reaction.