An acid protease from the broth of a 24 h cultivated Aspergillus niger BCRC 32720 was purified to electrophoretical homogeneity by CM Sepharose FF and Sephacryl S-100 HR chromatographs. The specific activity, purification fold, and yield were 23.29 kU/mg, 2.5, and 24.2%, respectively. Molecular mass (M) and N-terminal amino acid sequence were 47.5 kDa and SKGSAVTT, whereas the pH and temperature optima were at 2.5 and 50 °C, respectively. It was stable at pH 2.0-4.0 or ≤40 °C and activated by Fe(2+) and cysteine, but partially inhibited by phenylmethanesulfonyl fluoride and tosyllysine chloromethyl ketone and highly inhibited by Ag(+), Sn(2+), Fe(3+), Sb(3+), and pepstatin A. It was considered to be an aspartic protease.