Abstract 1. 1. The synthesis of medium-chain fatty acids by cell-free extracts of rabbit and rat mammary glands in proportions similar to those synthesized in vivo only occurred at high protein concentrations. The inability of subcellular fractions of guinea-pig mammary gland to synthesize medium-chain fatty acids correlates with the presence of only long-chain fatty acids in guinea-pig milk fat. 2. 2. Dilution of subcellular fractions progressively increased the proportion of long-chain fatty acids synthesized until the pattern resembled that obtained with isolated fatty acid synthetase. 3. 3. The specificity of fatty acid synthesis was not influenced by the esterification of the synthesized fatty acids into glycerides. 4. 4. Fatty acid synthetase may interact with other protein components in the particle-free supernatant fraction of rabbit and rat mammary gland, thereby altering its chain-length specificity.