A methylotrophic yeast, Candida boidinii, exhibits D-amino acid oxidase activity (DAO, EC 1.4.3.3) during its growth on D-alanine as a sole carbon or a nitrogen source. The structural gene (DAO1), encoding DAO, was cloned from a genomic library of C. boidinii, The 1035-bp gene encoded 345 amino acids and the predicted amino acid sequence showed significant similarity to those of DAOs from other organisms. The DAO1 gene was disrupted in the C, boidinii genome by one-step gene disruption. The DAO1-deleted strain did not grow on D-alanine as a carbon source but did grow on D-alanine as a sole nitrogen source (with glucose as the carbon source). These results suggested that, white DAO is critically involved in growth on D-alanine as a carbon source, there should be another enzyme system which metabolizes D-alanine as a nitrogen source in C, boidinii, We also showed that the three C-terminal amino acid sequence of DAO, - AKL was necessary and sufficient for the import of DAO into peroxisomes. Copyright (C) 2000 John Wiley & Sons, Ltd.