Cysteinyl dipeptidase from A. oryzae (CdpA) was produced in Escherichia coli and purified. CdpA formed a homodimer and its molecular mass was determined as 109 kDa. CdpA-specific activity to Cys-Gly was 3.04 U/mg. The enzyme showed maximum hydrolyzing activity toward Ala-Cys, followed by Leu-Cys, Cys-Gly, Cys-Ala, and Gly-Cys among the cysteine-containing dipeptide substrates. Its substrate specificity was distinct from those of other cysteinyl dipeptidases of the M20 family. The activity of CdpA was increased by addition of Mn2+, Zn2+, and Co2+ at 0.1 mM. The activity was inhibited in the presence of Fe2+. Several protease inhibitors reduced the activity. The complete inhibition of enzyme activity by EDTA indicates that CdpA is a metallopeptidase. It was optimally active at pH 7-8 and stable at pH 6-9 and at up to 40 "¢Å½. This study was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).