The cytosol from lactating-rabbit mammary gland contains a medium-chain acyl-thioester hydrolase. This hydrolase terminates chain lengthening of the fatty acids synthesised by fatty acid synthetase so as to release C8:0 and C10:0 fatty acids which are characteristic of rabbit milk. The medium-chain hydrolase and the fatty acid synthetase present in this cytosol have been shown to be immunologically distinct. When fatty acid synthetase was purified from this cytosol it showed unexpected immunological reactivity towards antiserum raised to the medium-chain hydrolase. The precipitate formed was not due to fatty acid synthetase, but to medium-chain hydrolase contaminating the synthetase. However, the proportion of this medium-chain hydrolase which was recovered with the purified synthetase was too small to be detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and was too small to elicit an antibody response in sheep. Immunological techniques have shown that the medium-chain hydrolase appears in rabbit mammary gland between days 17 and 22 of pregnancy. This coincides with the onset of milk-fat synthesis. The medium-chain hydrolase could not be detected in the cytosol from lactating-rabbit liver.