An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position.