The biosynthesis of polyketomycin was investigated by feeding ¹³C-labeled acetate and propionate to the growing cultures of Streptomyces diastatochromogenes Tü 6028. ¹³C NMR spectral analysis demonstrated the polyketide origin of the aglycone and the dimethylsalicyloyl moieties. The O-methyl group and 6-CH3 of the aglycone as well as 3B-CH₃ of L-axenose and 3C-CH₃ of the salicyloyl residue were labeled by feeding L-[methyl-¹³C]methionine. Both deoxysugars emerged from D-glucose. The biosynthesis of the aglycone and the assembly of the glycoside are discussed. The polyketomycin producing strain may be a candidate for further exploration in combinatorial biosynthesis.