The biosynthetic origins of the carbon skeleton of milbemycins alpha 2, alpha 4 and D were studied. 13C Labeled antibiotics, milbemycins alpha 2, alpha 4 and D, were isolated from the culture broth of Streptomyces hygroscopicus subsp. aureolacrimosus strain Au-3 after feeding [1-13C]acetate, [1-13C]-propionate, [3-13C]propionate, [1-13C]isobutyrate, DL-[2-13C]valine and L-[methyl-13C]methionine, and 13C NMR spectra of the antibiotics thus obtained were measured. It was revealed that the carbon skeleton, except for carbon 25, of milbemycins alpha 2, alpha 4 and D are derived from seven acetate units and five propionate units. It was also shown that the methyl, ethyl and isopropyl groups at carbon 25 in milbemycins alpha 2, alpha 4 and D are derived from acetate, propionate and isobutyrate or DL-valine, respectively, and the methyl carbon of the methoxy group at carbon 5 in milbemycins alpha 2 and alpha 4 was enriched by L-[methyl-13C]methionine.