Biosynthetic incorporation experiments were performed with carbon-13 labelled precursors including sodium [1-13C]-, [2-13C], and [1,2-13C2]-acetate ar well as [methyl-13C] methionine into antibiotic F-244 (1) in growing cultures of Fusarium sp. ATCC 20788. After conversion to the methyl ester 2, analysis by NMR showed that the carbon skeleton of 1 derives from seven intact acetate units; the remaining four carbons are from methionine. Hence, the pathway is similar to that reported for 1 in Scopulariopsis. The biogenesis of the hydrogen atoms in 1 was also investigated. Incorporation of sodium [1-13C,18O2]acetate gives 2, which exhibits 18O-induced isotope shifts at C-1 and C-3. The labelling pattern is consistent with formation of the β-lactone ring by nucleophilic attack of a C-3 hydroxyl group in the nascent polyketide precursor onto the C-1 carbonyl.