Twelve new milbemycins have been isolated and characterized from some strains derived from Streptomyces hygroscopicus subsp. aureolacrimosus SANK60286 and SANK60526. The metabolites 1-4 and 9-ll were produced by strain RM28D-688 SANK60797 as minor products. The metabolites 5-8 were obtained from a broth of strain 57-338 SANK61796. Strain MK-1391SANK62896 was used for the production of metabolite 12. The new metabolites, eight a-class and four /3-class compounds, have newstructural features. For example, milbemycins a26 and a27, have the 26-hydroxy moiety, and other derivatives (milbemycins a20_23) have different side chains at the C-26 position from those of milbemycins an and au. In addition, 5-hydroxylmilbemycin /37 (j312), involved in the major biosynthetic pathway of 25-methyl and 25-ethyl milbemycins, was discovered.