A method to determine intracellular cation contents in Dunaliella by separation on cation-exchange minicolumns is described. The separation efficiency of cells from extracellular cations is over 99.9%; the procedure causes no apparent perturbation to the cells and can be applied to measure both fluxes and internal content of any desired cation. Using this technique it is demonstrated that the intracellular averaged Na(+), K(+), and Ca(2+) concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K(+), and 0.3 millimolar Ca(2+) are 20 to 100 millimolar, 150 to 250 millimolar, and 1 to 3 millimolar, respectively. The intracellular K(+) concentration is maintained constant over a wide range of media K(+) concentrations (0.5-10 millimolar), leading to a ratio of K(+) in the cells to K(+) in the medium of 10 to 1,000. Severe limitation of external K(+), induces loss of K(+) and increase in Na(+) inside the cells. The results suggest that Dunaliella cells possess efficient mechanisms to eliminate Na(+) and accumulate K(+) and that intracellular Na(+) and K(+) concentrations are carefully regulated. The contribution of the intracellular Na(+) and K(+) salts to the total osmotic pressure of cells grown at 1 to 4 molar NaCl, is 5 to 20%.