Feeding experiments with [1-C-13]-, [1,2-C-13(2)]-, and [1-C-13,O-18(2)]acetate as well as a fermentation under an O-18-enriched atmosphere resulted in information regarding the biosynthetic origin of all carbon and of the oxygen atoms of the landomycin A aglycon moiety as well as of the direction of the incorporation of the decaketide chain. Only two of the six oxygens in landomycinone seem to originate from the acetate building blocks. This raises questions about current hypotheses for the formation of multicyclic aromatic polyketides. Some unusual prearomatic deoxygenation steps early in the biosynthesis have been proposed. As an additional result of the biosynthetic investigations, the structure of landomycin A and thus of all other landomycins were revised concerning the position of the phenol-glycosidically linked deoxysaccharide chain from 2 to 1.