The influence of several parameters of the incubation medium: tonicity, hydrogen ion concentration, and concentration of inorganic phosphate, on the phospholipid composition of extracts from rat liver mitochondria, produced under conditions of the usual extraction, as well as in the case of extraction with strongly acidified mixtures of solvents. It was established that after the usual extraction with a mixture of chloroform and methanol, 8-25% phospholipids can be additionally removed from the mitochondria by extraction with acidified mixtures of solvents. It was shown that the amount of difficult-to-extract lipids depends on the tonicity and ionic composition of the mitochondrial incubation medium. It is suggested that two lipid pools exist in the mitochondria: tightly bound and labile. The position of equilibrium between the tightly bound and labile lipids is effectively regulated by the tonicity and other external factors.