Abstract Biosynthetic origin of macrolactam polyketide antibiotic cremimycin was investigated by feeding experiments with [1-13C]acetate, [1,2-13C2]acetate, [1-13C]propionate, succinate-d4, and d -[6,6-2H2]glucose. NMR analysis of the resultant isotope-enriched cremimycins showed distinctive incorporation patterns, which suggested that the aglycon of cremimycin was constructed from two propionates and eleven acetates. Thus, 3-oxononanoate was proposed as a potential polyketide intermediate, that is, aminated to be the unique nitrogen-containing moiety of cremymicin. Further, characteristic propionate biosynthetic pathway in the cremimycin-producing strain was also described.