AbstractA cell‐free system, consisting of particle‐free rat liver cytoplasm plus purified mitochondria, was used to study the synthesis of fatty acid from14C‐U‐alanine. Incorporation of label into lipid was dependent upon the presence of both cytoplasm and mitochondria. However, the metabolic state of the animal, prior to sacrifice, determined the capacity of various mitochondrial preparations to reconstitute the system. Mitochondria prepared from the livers of starved, fat‐fed, or alloxan‐diabetic rats failed to support fatty acid synthesis to the same extent as mitochondria prepared from control animals. Likewise, less14CO2 was produced in the system reconstituted with liver mitochondria prepared from starved rats. Possible mechanisms are discussed.