Biosynthesis of hibarimicin was studied based on the feeding experiments with 13C labeled acetates. All carbons in the aglycon, except for the methoxy carbons, were derived from acetate. The carbon framework of the aglycon was proved to be constructed by dimerization of an intermediate which was biosynthesized via the decarboxylation and skeltal rearrangement starting from an undecaketide. The rearrangement was confirmed by detecting the long range (three-bond) coupling between two carbons in the difference spectra of selective 13C decoupled INADEQUATE of hibarimicin B labeled with sodium [1,2-13C2] acetate.