1. Purified cow mammary gland fatty acid synthetase synthesized long-chain unesterified and short-chain esterified fatty acids. 2. A direct relationship was observed between the amount of short-chain products synthesized and the concentration of acetyl-CoA in the incubation medium. 3. The short-chain products were identified as butyryl-CoA and hexanoyl-CoA. 4. Inhibition of the terminating thioester hydrolase of the fatty acid synthetase complex with phenylmethanesulphonyl fluoride did not inhibit the synthesis of short-chain products. 5. It is suggested that the synthesis of short-chain fatty acids involves the reverse of the ‘loading’ reaction.

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