D-Amino acid oxidase (DAAO) is an FAD-dependent enzyme that metabolizes D-amino acids in microbes and animals. However, such ability has not been identified in plants so far. We predicted a complete DAAO coding sequence consisting of 1158 bp and encoding a protein of 386 amino acids. We cloned this sequence from the leaf cDNA population of maize plants that could utilize D-alanine as a nitrogen source and grow normally on media containing D-Ala at the concentrations of 100 and 1000 ppm. For more understanding of DAAO ability in maize plant, we produced a recombinant plasmid by the insertion of isolated cDNA into the pMALc2X Escherichia coli expression vector, downstream of the maltose-binding protein coding sequence. The pMALc2X-DAAO vector was used to transform the TB1 strain of E. coli cells. Under normal growth conditions, fused DAAO (with molecular weight of about 78 kDa) was expressed up to 5 mg/liter of bacterial cells. The expressed product was purified by affinity chromatography and subjected to in vitro DAAO activity assay in the presence of five different D-amino acids. Fused DAAO could oxidize D-alanine and D-aspartate, but not D-leucine, D-isoleucine, and D-serine. The cDNA sequence reported in this paper has been submitted to EMBL databases under accession number AM407717.