Adaptation of Staphylococcus aureus to a change in salinity was studied by estimating the intracellular content of water and proline after alteration of the salt concentration of the culture medium. The intracellular water content of S. aureus cultured in normal broth was 1.70 g/g (dry weight). After transfer to 1.8 M NaCl-containing broth, the water content decreased to 0.80 g/g (dry weight) within 1 min. After changing the salt concentration of the medium, intracellular free proline (assumed to be one of the osmoregulators in S. aureus) increased gradually from 0 to 1,400 mumol/g (dry weight) during 30 min of incubation at 37 degrees C. The water content rose to 0.88 g/g (dry weight) in 30 min. Proline was not taken up at 0 to 4 degrees C, suggesting that the process was one of active transport. The salt tolerance of S. aureus, therefore, appears to occur initially by dehydration of the cell after transfer from a medium of low salinity to one of high salinity and then by accumulation of proline, which carries water into the cell with it.