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D-amino acid oxidase (DAO, EC 220.127.116.11) is one of the flavoenzymes in peroxisomes catalyzing the oxidation of D-amino acids to α-imino acids. By reverse transcription-polymerase chain reaction, DAO cDNA was cloned from the mRNA of zebrafish embryos. The deduced zebrafish DAO amino acid sequence revealed a 348-amino acid polypeptide containing two FAD-binding sites (amino acids positions 7G to 12G, and 236V to 247W), and a peroxisomal targeting signal sequence (346Ser-347Arg-348Leu) at the C-terminal end. Following comparison of the sequences, we found that the zebrafish DAO polypeptide shared the sequence identities of 93%, 79%, 77%, 77%, 46%, 43% and 25% of the reported DAO of carp, human, mouse, pig, Streptomyces coelicor, Rhodotorula gracilis and Schizosaccharomyces pombe, respectively. Whole-mount in situ hybridization revealed that dao was a maternally inherited gene and that its expression was first observed in the zygote period, gradually down-regulated from the cleavage period to the early blastula stages, and had declined to an undetectable level at 6 h post-fertilization (hpf). At 18-hpf, the zebrafish dao transcripts were detected in a very weak manner, and their expression was restricted in the future head and brain regions as well as in the yolk syncytial layers of the yolk and yolk extension regions. At later stages, dao expression was detected in the brain, gut, swimming bladder, pronephric duct, optic tectum, ventrolateral tract and in the floor plate of the developing spinal cord. The enzymatic activities of DAO were also examined and showed that the DAO enzyme activities were expressed in a dynamic and stage-dependent manner: first it was detected at the 1-cell stage at an extremely low level (0.007 ± 0.005 U/mg), reached the maximum amount at 6 days post-fertilization (dpf) (0.323 ± 0.04 U/mg), and gradually down-regulated to the basal level (0.019 ± 0.005 U/mg) in a 1-month juvenile. From these observations, we conclude that zebrafish dao is a maternally inherited gene, and that its expression was restricted in some neurons, gut, and pronephros and could be used as a good marker for the study of pronephridial development.