Abstract In the 250- to 300-nm ultraviolet region, purified ascorbate oxidase exhibits a strong absorption band at 280 nm, having a distinct shoulder absorption at 290 nm. The E1%1 cm (280 nm) value has been found to be 21.7 ± 1.2. In the visible light region, 300 to 1000 nm, ascorbate oxidase exhibits five absorption bands, i.e. at 330, 460, 610, 770, and 880 nm. The extinction coeffients at each wave length have been found to be 2000, 390, 1300, 600, and 560 per g atom copper per liter per cm, respectively. The ratio A280 nm:A610 nm has been found to be 25.6. Purified ascorbate oxidase exhibits a maximal emission spectrum at 325 nm and the fluorescence quantum yield has been calculated to be 0.06. The circular dichroism of ascorbate oxidase in the spectral range of 185 to 240 nm indicates that in 0.01 m phosphate buffer, pH 7.6, ascorbate oxidase exists predominantly in the antiparallel β conformation. As revealed by CD there are at least four transitions in the absorption of the copper chromophore in the spectral range 300 to 700 nm. The absorption and CD spectra of ascorbate oxidase have been examined and compared with those of other copper proteins, and the results support the view that the copper of ascorbate oxidase exists in complex forms. The electron paramagnetic resonance spectrum of ascorbate oxidase is characterized by extremely small low field hyperfine splittings. The gm, g||, and |A| values have been found to be 2.074, 2.244, and 0.006 cm-1, respectively. These and other spectral characteristics of ascorbate oxidase have been found to be very similar to those of other copper proteins particularly ceruloplasmin and laccase, but are distinct from small molecule cupric complexes. The absorption and CD spectra of ascorbate oxidase in the range 400 to 700 nm have been compared and found to be similar to those that have been reported for the model copper (II)-histidine-containing peptide complexes. The spectral data suggest that the copper of ascorbate oxidase is in an environment of low symmetry involving nitrogen atom ligands.