Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.