Glycolate oxidase is a flavin-dependent enzyme that catalyzes the oxidation of α-hydroxy acids to the corresponding α-keto acids, with reduction of molecular oxygen to hydrogen peroxide. A number of probes have been used to investigate the oxidative half-reaction catalyzed by the enzyme, including steady state and rapid kinetics, pH studies, solvent kinetic isotope effects, and solvent viscosity effects. Here we present the first spectroscopic evidence of the formation of an intermediate with absorbance features resembling those of a flavosemiquinone in the oxidative half-reaction of glycolate oxidase.