We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Nn.' from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.Halophilic bacteria belong to two major kingdoms. The archaebacterial extreme halophiles generally require 2 to 3 M NaCl and can grow in saturated brines (16,25). The composition of their cell envelopes differs from that of eubacterial cells, and they have high proportions of negatively charged proteins in their membranes, ribosomes, and other cell components. They are known to accumulate intracellular potassium ions to balance the extracellular sodium concentrations (16,25).The second group, the eubacterial halophiles, are far more widely distributed, both environmentally and among families and genera (19,22,32), and they are of great ecological and economic importance in agriculture, food industry, and other more specialized processes. The eubacterial halophiles also show more wide-ranging salt requirements and tolerances, i.e., they are more adaptable. The mechanisms and components which enable them to grow over such a wide range (from 3-to 10-fold, according to species) of salt concentrations are still largely unknown. One of the most important questions still unresolved involves the true intracellular ion concentrations relative to those in the medium and how the organisms deal with the osmotic and ionic gradients across their cell membranes. This problem has been highlighted by studies which showed that in vitro protein-synthesizing systems from such organisms (35) and most of their intracellular enzymes were inhibited by NaCl concentrations at which the cells had been grown (12,17,35). On the other hand, earlier studies of intracellular salt concentrations in such organisms suggested that the total concentration of intracellular cations was more or less equal to the cation concentration in the extracellular milieu (3,5,15,20,23,27,28,33