Strain Au-3, a mutant of Streptomyces hygroscopicus ssp. aureolacrimosus, obtained with UV irradiation, was a high-yield strain of milbemycins D, E, F, G and H. Fermentation studies on the strains were conducted in shake flasks and 30 l jar fermentors. Isolation of the metabolites was performed by adsorption on resinous adsorbent followed by elution with aqueous methanol and separated by silica gel column chromatography. Milbemycin D was obtained as colorless needles after recrystallization; milbemycins E, F, G and H were purified to homogeneity by column chromatography. Physicochemical characterization revealed that milbemycins D, E, F, G and H were new antibiotics possessing the 16-membered macrocyclic lactone with a 6,6-membered spiroketal ring system.