The phospholipid composition of Rhodobacter sphaeroides cells resuspended in varioushypertonic solutions has been examined by thin-layer chromatography and ESI mass spectrometry. R.sphaeroides responds to hyperosmotic stress by increasing the amount of cardiolipin in the membranes;this phenomenon occurs in spheroplasts also. Cardiolipin increases quickly and continuously during thetime when the cells are resuspended in hypertonic medium. The optimum of stimulation of the neosynthesisof cardiolipin during osmotic stress was found to be at external 1 osm. ESI-MS analyses allowed theidentification of two different cardiolipins in R. sphaeroides: the tetravaccenylcardiolipin ([M − H]-,m/z 1456.9) and the trivaccenylmonopalmitoylcardiolipin ([M − H]-, m/z 1430.0).