In the present theoretical study, the active site of mammalian glutathione peroxidase (GPx) has been refined by including protein/active site interactions using the two-layer ONIOM(QM:MM) method. In this study, a full model consisting of 3113 atoms (with 86 active site atoms in the quantum mechanical (QM) region and the rest in the molecular mechanics (MM) region) has been constructed on the basis of the 2.9 Angstrom resolution X-ray structure of mammalian GPx. The "active site only" models give much larger root-mean-square (rms) deviations from the X-ray structure than the full model, indicating the importance of the protein environment on the structure of the active site. The still substantial rms errors of the optimized structures can be improved only when the full model complemented with two water molecules is considered, clearly indicating the existence of two missing water molecules at the active site of the mammalian GPx, which could be critical for the catalytic activity.