In the resting oxidized state (the fully oxidized "as-isolated" state) of cytochrome c oxidase (CcO) preparation, a resonance Raman band is observed at 755 cm(-1) upon 647.1 nm excitation in resonance with an absorption band at 655 nm. Addition of cyanide eliminates the Raman band concomitant with loss of the absorption band at 655 nm. These results strongly suggest that the Raman band at 755 cm(-1) originates from the O-O stretching mode of the bridging peroxide (Fe-O(-)-O(-)-Cu) in the O(2) reduction site of the fully oxidized "as-isolated" CcO. Although the peroxide bridged structure has been proposed on the basis of X-ray crystallography and reductive titration experiments, the present vibrational spectroscopic analyses reveal conclusively the chemical nature of the bridging ligand at the O(2) reduction site of the fully oxidized "as-isolated" bovine heart CcO.