The concentration of Na+ and K+ and the pH in the cytoplasm of Lettré cells was measured by monitoring the net flux of H+, Na+, or K+ across the plasma membrane which had been rendered permeable to these ions by the action of Sendai virus. Ion flux was measured directly by analysis of cell composition, or indirectly by observing the change in membrane potential of cells treated with a specific ionophore. Cytoplasmic concentrations of cations were obtained by establishing the concentration of the cation in the medium at which addition of Sendai virus causes no change in cytoplasmic cation content. The value of Lettré-cell pH was confirmed by direct measurement employing 31P nuclear magnetic resonance, and the values of Na+ and K+ concentration were confirmed by analysis of cell cation and water content. Lettré cells suspended at 32 degrees C in Hepes-buffered saline at pH 7.3 maintain a cytosolic pH of 7.0 and contain 50 mM Na+ and 80 mM K+.