Abstract Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants. Here we report the heterologous expression of glycolate oxidase in Escherichia coliand an isolation procedure which results in 4 mg pure protein per gram cell paste in only 1.5 days. This corresponds to a more than 50-fold improvement in yield compared to previously reported expression systems. The purified recombinant protein can be crystallized easily, and the crystals are isomorphous to those obtained from the protein isolated from spinach. The availability of large amounts of enzyme will be a great advantage in the 3D structural and biochemical studies of mutants and inhibitor complexes.