The milbemycins are the metabolites produced by Streptomyces B-41-146 strain and exhibit a remarkable pesticidal activity against mites such as two-spotted spider mite and citrus red mite, and insects such as rice leaf beetle and tent caterpillar, without phytotoxicity on many variety of crops at effective dosages. Milbemycin α3 and β1 were converted into the urethane derivatives containing a heavy atom, and the structures of which were determined independently by X-ray analysis. On the basis of the established structures of M.α3 and M.β1, those of the other constituents were determined by the chemical correlation and/or the comparison of spectral data (IR, UV,^1H ε ^<13>C NMR and Mass). The structures consist of three basic units: 16-membered lactone, substituted cyclohexene, and spiro ketal system containing two six-membered rings.