Limited digestion, with trypsin, of the fatty acid synthetase from rat mammary gland releases an enzymically active thioesterase component that, under denaturing conditions, consists of two major species of mol.wts. 35000 and 17500 and a minor species, mol.wt. 15,000. The 17500- and 150000-mol.wt. species are shown to originate from the 35000-mol.wt. species as a result of nicking by trypsin. The nicked polypeptides are enzymically active. The fatty acid synthetase is inhibited by [1,3-14C]di-isopropyl phosphorofluoridate, which is shown to bind to, and inactivate, two thioesterase active sites. When the [1,3-14C]di-isopropyl phosphate-labelled fatty acid synthetase is subjected to limited digestion with trypsin, all of the radioactivity is recovered in the isolated thioesterase component, i.e. in the 35000-mol.wt. polypeptide and its nicked products. Since the isolated thioesterase is shown to bind only one di-isopropyl phosphate residue per 35000-mol.wt. polypeptide, we conclude that the fatty acid synthetase has two thioesterase domains, both of which are removed by limited trypsin treatment.