A novel heme-containing ascorbate oxidase isolated from oyster mushroom, Pleurotus ostreatus, catalyzes oxidation of ascorbic acid (Kim et al., 1996). In this report, we describe the identification of intracellular substrates of the enzyme in the mushroom. Six compounds, which can serve as substrate of the heme-containing ascorbate oxidase, were identified as L-ascorbic acid, D-erythroascorbic acid, 5-O-(alpha-D-glucopyranosyl)-D-erythroascorbic acid, 5-O-(alpha-D-xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-O-(alpha-D-gluco-pyranosyl)-D-erythroascorbic acid, and 5-methyl-5-O-(alpha-D-xylopyranosyl)-D-erythroascorbic acid. All of the compounds were oxidized at a significant rate by the heme-containing ascorbate oxidase. Oxidation of the compounds produced equimolar amounts of hydrogen peroxide per mole of substrate.