Recently, we reported the development of a sensitive continuous spectrophotometric assay for the ascorbate-dependent mammalian enzyme dopamine beta-monooxygenase based on the novel chromophoric electron donor 2-aminoascorbic acid [K. Wimalasena and D.S. Wimalasena (1991) Anal. Biochem. 197, 353-361]. We now report that ascorbate oxidase (EC 1.10.3.3, L-ascorbate:O2 oxidoreductase) also catalyzes the oxidation of 2-aminoascorbic acid to chromophoric 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment). The reaction is kinetically well behaved, displaying the expected stoichiometry for an oxidase-catalyzed reaction with respect to oxygen and the oxidation product (red pigment), demonstrating that 2-aminoascorbic acid is a well-behaved alternative substrate for the enzyme. Ascorbate oxidase is a very efficient enzyme toward its natural substrate, ascorbic acid. Although 2-amino-ascorbic acid is a significantly weak substrate for the enzyme in comparison to ascorbic acid, as indicated by the apparent initial rate kinetic parameters, the high extinction coefficient of the red pigment under our assay conditions suggests that this novel reactivity of the enzyme could be used to design a sensitive, convenient, and continuous spectrophotometric assay for ascorbate oxidase. While this assay is more convenient than the existing oxygen monitor assay, its adaptability to measure the activity of the enzyme in the immobilized form may be helpful in the development of technologies for the automated detection of ascorbic acid in biological fluids for industrial or clinical applications. In addition, this novel reactivity of the enzyme may be used to examine the substrate specificity and the mechanism of action of the enzyme.