A Na+ uptake-associated vacuolar alkalinization was observed in roots of two barley cultivars (Arivat and the more salt-tolerant California Mariout) by using 23Na and 31P in vivo NMR spectroscopy. A NaCl uptake-associated broadening was also noted for both vacuolar Pi and intracellular Na NMR peaks, consistent with Na+ uptake into the same compartment as the vacuolar Pi. A close coupling of Na+ with H+ transport (presumably the Na+/H+ antiport) in vivo was evidenced by qualitative and quantitative correlations between Na+ accumulation and vacuolar alkalinization for both cultivars. Prolongation of the low NaCl pretreatment (30 mM) increased the activity of the putative antiport in Arivat but reduced it in California Mariout. This putative antiport also showed a dependence on NaCl concentration for California Mariout but not for Arivat. No cytoplasmic acidification accompanied the antiporter activity for either cultivar. The response of adenosine phosphates indicated that ATP utilization exceeded the capacity for ATP synthesis in Arivat, but the two processes seemed balanced in California Mariout. These comparisons provide clues to the role of the tonoplast Na+/H+ antiport and compensatory cytoplasmic adjustments including pH, osmolytes, and energy phosphates in governing the different salt tolerance of the two cultivars.