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 Ca2 concentrations in Dunaliella salina cultured at 1 to 4 molar NaCl, 5 millimolar K@, and 0.3 millimolar Ca2" are 20 to 100 millimolar, 150 to 250 millimolar, and I 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 linlitation 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%.The unicellular green alga Dunaliella has the remarkable capacity to grow and adapt itself to media ranging in salinity from about 50 mM up to 5 M NaCl (4). It has been clearly established that the major means of osmoregulation of this wall-less alga is by production of internal glycerol at concentrations which are proportional to the external NaCl concentration (5). However, there is a considerable disagreement as to the contribution of inorganic ions, in particular for Na+ to the overall osmotic pressure inside the cells. Different groups have determined intracellular Na+ concentrations ranging from around 50% (12,16), to less than 10% (3,6,15) and even to about 1% (8) of the external NaCl concentration. These differences seem to be mainly due to difficulties in effecting a complete separation of the cells from the extracellular Na+; to errors in the determination of intracellular osmotic volumes with different extracellular markers (8,12,14,15) MATERIALS AND METHODS Growth Conditions. D. salina were grown in batch cultures, in media containing 1 to 4 M NaCl, 50 mm NaHCO3, 5 mM KNO3, 5 mM MgSO4, 0.3 mM CaCI2, 0.2 mM KH2PO4, 1.5 /LM FeC13,6 ,uM EDTA, 185 ,uM H3BO4, 7 AM MnC12, 0.8 piM ZnC12, 0.02 AM CoCl2, and 0.2 nm CuCl2. Cultures were grown under continuous illimination with white fluorescent lamps at 3,800 lux, 26°C with slow continuous shaking and kept at the logarithmic growth phase. Adaptations to different NaCl concentrations were made by growth for several days in 1 to 4 M NaCl. For adaptation to low K+ concentrations the cells were grown for several days in a K+ deficient medium followed by 48 to 72 h growth with the specified K+ concentrations. For steady state distribution of 22Na+, 86Rb+, or 45Ca2 , algae were cultured for 24 to 48 h in the presence of 5 to 50 ACi/ml 22Na+ or 0.1 u...