The milbemycins, a group of potent, broad-spectrum antiparasitic and pesticidal agents, are architecturally novel antibiotics of 16-membered macrocyclic lactone. Seven new milbemycin analogues designated as milbemycins D, E, F, G, H, J and K were isolated from the fermentation broth of the mutant strain of Streptomyces hygroscopicus subsp. aureolacrimosus. The structural determination of these new components was made mainly by comparing with mass spectra, and 114 and 13C NMR spectra of milbemycin a-and (9-series previously published from our laboratory. Milbemycins D, E, F, G and H have characteristically an isopropyl side chain at C-25 which differs from the known milbemycin family bearing methyl or ethyl group at C-25. Milbemycins J and K possess a ketone group at C-5 instead of a hydroxyl or methoxy group.Apart from X-ray crystallography, the R-configuration of the hydroxyl group at C-5 could be best explained both by application of CD allylic benzoate method to the p-N,N-dimethylaminobenzoate of milbemycin D and by comparison of the specific rotation of milbemycin D itself and its acetate with the epimeric isomers at C-5. Milbemycins1-3), which was found and structurally elucidated in 1975 in our laboratory as shown in Fig. 1, is a group of the fermentation products of Streptomyces hygroscopicus subsp. aureolacrimosus with potent, broad-spectrum anthelmintic, insecticidal and acaricidal activity, but is devoid of the antibacterial activity associated with macrolide derivatives. Common characteristic feature of the milbemycin is the presence of both a 6,6-membered spiroketal and a cyclohexene ring fused to the 16-membered macrocyclic lactone ring. Milbemycins can be divided structurally into two groups of a-and Qseries: a-series has the tetrahydrofuran ring fused to the cyclohexene ring, (l-series has no tetrahydrofuran ring. After several years of our finding milbemycins, the Merck group reported on the avermectins, isolated from the fermentation broth of Streptomyces avermitilis, which were glycosides with an a-L-oleandosyl-a-L-oleandosyl moiety at C-13 of milbemycin, and showed a similar biological activity to that of the milbemycin4,5)In the course of our fermentation development, novel milbemycin analogues designated as milbemycins D, E, F, G, H, J and K were obtained from the fermentation broth of the mutant strain of S. hygroscopicus subsp. aureolacrimosus. The fermentation, isolation and physico-chemical properties of these new metabolites have been reported in the previous papers"').In this paper we wish to report the structure determination of these new metabolites and the absolute configuration of milbemycin D.