The biosynthesis of antibiotic 1233A (F-244) was studied by feeding ¹³C-labeled precursors to the producing organism, Scopulariopsis sp. F-244. ¹³C NMR spectroscopy established that 1233A is derived from 4 methionines and 7 acetates. Seven acetates are condensed to form a hexaketide and 4 methyl residues from methionine are introduced into the main skeleton. The β-lactone is derived from the α-carboxylic acid of the hexaketide. Since methionine was efficiently incorporated into 1233A, radiolabeled 1233A was prepared biosynthetically by feeding [¹⁴C]methionine to the producer. As a result, [¹⁴C]1233A was obtained with high specific radioactivity (27.2 μCi/μmiol).