The biosynthetic origins of the Ca 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 aureolacrimosus strain Au-3 after feeding [1-13C]acetate, [1-13C]propionate, [3-13C]propionate, [1-13C]isobutyrate, DL-[2-13]valine and L-[methyl-13C]methionine, and 13C NMR spectra of the antibiotics thus obtained were measured. The carbon skeleton, except for C25, of milbemycins .alpha.2, .alpha.4 and D are derived from 7 acetate units and 5 propionate units. The methyl, ethyl and isopropyl groups at C25 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.