D-Aspartate oxidase (DDO) cDNAs were isolated from the human brain RNA using the RT-PCR method. Two forms (DDO-1 and DDO-2) of DDO mRNA were detected. Structural analysis of the DDO cDNAs and genomic DNA showed that DDO-1 and DDO-2 are produced by alternative splicing from a single gene. A protein encoded by the DDO-1 cDNA consists of 341 amino acids, and the amino acid sequence of DDO-2 was identical to that of DDO-1 except for the absence of 59 amino acids covering residues 95-153 of DDO-1. A homogenous preparation of DDO-1 was obtained using an expression system in Escherichia coli. DDO-1 selectively catalyzed the oxidative deamination of D-aspartate and its N-methylated derivative, N-methyl D-aspartate; the values of K(m) and k(cat) for D-aspartate were 2.7 mM and 52.5 mol D-aspartate oxidized x s(-1) x mol(-1) and those for N-methyl D-aspartate were 6.8 mM and 37.7 mol N-methyl D-aspartate oxidized x s(-1) x mol(-1), respectively.