Cerulenin, (2R)(3S)-2,3-epoxy-4-oxo-E,E'-7,10 dodecadienoylamide, is a potent inhibitor of fatty acid synthase systems isolated from various microorganisms and animal tissues.1} This antibiotic specifically blocks the activity of 2-oxoacyl thioester synthase (condensing enzyme).1} We have proposed that cerulenin covalently binds to the cysteine-SH at the active center of the condensing enzyme.2,3) Cerulenin has also been found to inhibit the biosynthesis of various polyketide-derived secondary metabolites such as macrolides4) and nanaomycin.5) Because of its unique mode of action and usefulness as a biological reagent, attempts were made to prepare [3H]- and [14C]cerulenin by the present authors6,7) and other research groups.8) Labeled cerulenin has been used for the binding and active-site studies on fatty acid synthase.6-8) However, the specific radioactivities of these preparations were not very high (0.46 and 0.016 mCi/mmol of [3H]cerulenin)7) although Tsukamoto et al.8) reported on [14C]cerulenin with fairly high specific radioactivity (2.21 mCi/mmol). On the other hand, Robert and Leadlay9,10) prepared [3H]tetrahydrocerulenin (45 Ci/mmol) by chemical reduction of cerulenin with tritium gas over PtO2. The purpose of this paper is to present a method of biosynthetic preparation of labeled cerulenin with high specific radioactivity. This method yielded 13.2 mCi/mmol of [3H]cerulenin.