AtNHX1, a vacuolar Na(+)/H(+) antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na(+)/H(+) exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H(+)-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na(+)/H(+) exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na(+)/H(+) antiporter AtNHX1. V-ATPase and vacuolar Na(+)/H(+) antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na(+)/H(+) antiporter might be appropriate for producing plants with a higher salt tolerance ability.