Single crystals of cholesterol oxidase from Brevibacterium sterolicum containing a covalently bound form of the FAD cofactor have been obtained, The crystals are grown by vapor diffusion using the hanging drop technique from 12% polyethylene glycol, M(r) 8000, and 75 mM MnSO4 as the precipitant at pH 5.2, In order to obtain large diffraction quality crystals, nucleation must occur at 22 degrees C with subsequent growth at 17 degrees C, The crystals belong to the monoclinic space group P2(1) with cell dimensions a = 78.5 Angstrom, b = 126.7 Angstrom c = 82.4 Angstrom and beta = 108.9 degrees with two protein molecules per asymmetric unit, Diffraction of these crystals has been observed to at least 2.2 Angstrom resolution and they are suitable for an X-ray structure analysis. (C) 1996 Academic Press, Inc.