AbstractA method for isolation and quantitation of cellular free fatty acid has been developed. When this method was used to quantitate the free fatty acid content of various cells and tissues, their levels of free fatty acids were found to vary over a wide range. In comparing tissue culture cells having different levels of free fatty acid, it was demonstrated that the conditions of culture and the type of serum in the medium are not responsible for the difference in levels. Isotopic studies have shown that the cellular free fatty acid is not biosynthesized, but is derived from the free fatty acid of the medium. Preliminary studies on the fate of the intracellular free fatty acid and a discussion of possible factors controlling the level of this compound in cells are presented.