Abstract Linoleic acid-1-14C was incubated with subcellular fractions of rat liver to study the mechanism of polyunsaturated fatty acid synthesis. The most effective system for the chain elongation of linoleic acid to eicosadienoic acid was liver microsomes plus malonyl coenzyme A with cofactors, NADPH, and ATP. Under anaerobic conditions, the chain elongation reaction could be separated quantitatively from the dehydrogenation reaction which otherwise required the same system. The chain elongation of linoleic acid was inhibited by saturated fatty acids of which myristic and pentadecanoic acids were most effective. Mono-unsaturated and polyunsaturated fatty acids also act as inhibitors. From consideration of reaction kinetics and inhibition by other fatty acids, the conversion of linoleic acid to γ-linolenic acid seems preferred over the conversion of linoleic acid to homolinoleic acid.