Streptomyces fumanus, intramurally coded as culture LL-F42248, produces a series of pyrrolomycins including dioxapyrrolomycin (1) as the principal component. Our biosynthetic studies revealed that feeding labeled acetate to growing cultures of S. fumanus yielded pyrrolomycins labeled in the phenyl ring only. When l-[methyl-13C]methionine was fed, the labeled carbon atom was found in the methoxy group of pyrrolomycins H-J and in the methylenedioxy bridge of dioxapyrrolomycin. A Na15NO3-enriched medium was employed to produce 15N-labeled pyrrolomycins in which both nitrogen atoms were highly enriched, whereas feeding of 15N-labeled l-proline furnished pyrrolomycins labeled in the pyrrole moiety. Thus, S. fumanus elaborates the pyrrolomycin skeleton from proline and a polyketide precursor. Since the organism readily converted 13C- or 15N-labeled pyrrolomycin C, G, or H into the correspondingly labeled dioxapyrrolomycin, these minor pyrrolomycins are actually precursors of the ultimate product, dioxapyrrolomycin.