Differential scanning calorimetry (DSC) was used for determining the number of unfreezable water molecules per molecule of phosphatidylserine from bovine spinal cord (PS) or dimyristoyl phosphatidylserine (DMPS) and dimyristoyl phosphatidylcholine (DMPC), alone or in mixtures with cholesterol. It was assumed that the unfreezable water molecules are tightly bound to the phospholipid. It was found that when the phospholipids are in the gel state and in the absence of cholesterol, PS binds 2.5 water molecules, DMPS 3.8 water molecules and DMPC 3.5 water molecules. In the presence of cholesterol the number of water molecules bound increases in the region where phase separation of cholesterol takes place [D. Bach, Chem. Phys. Lipids 35 (1984) 385-392; E.J. Wachtel, N. Borochov, D. Bach, Biochim. Biophys. Acta 1066 (1991) 63-69; D. Bach, N. Borochov, E. Wacktel, Chem. Phys. Lipids, submitted].