Cholesterol oxidase (CO) is a flavoenzyme catalysing the oxidation and isomerisation of cholesterol. The reductive half reaction occurs via a two electron hydride transfer from the substrate to the FAD cofactor.  To better understand the mechanism of hydride transfer the single crystal neutron diffraction structures of the reduced and oxidised perdeuterated proteins have been pursued.   Toward this aim, the high resolution (1.12Å) X-ray structure of the reduced protonated enzyme was obtained.  The presence of the hydride transferred to the flavin during the reductive half reaction is visible in the difference density map at a distance of 1.13 Å from the N5 of the flavin.  However to definitively identify hydrogen atoms in the structure we require the neutron diffraction structure.   Previously, neutron diffraction data to 2.2Å resolution from crystals of the oxidised D₂O-exchanged enzyme were collected from the IMAGINE instrument at the High Flux Isotope Reactor (Oak Ridge National Laboratory).  The structure obtained reveals deuterium atoms for many exchangeable positions.  However, non-exchangeable hydrogen atoms suffer from significant density cancellation effects due to the nearby positive scattering carbon atoms.  To circumvent this problem we have successfully expressed, purified and crystallised the perdeuterated CO and obtained preliminary neutron diffraction data to 2.1Å resolution for the oxidised enzyme at Oak Ridge National Laboratory.   Progress toward the perdeuterated structure will be presented.