The aminopeptidase N (TH-4AP) of Streptomyces sp. TH-4 was purified from a culture supernatant. The purified enzyme had a molecular mass of 95 kDa. The gene encoding TH-4AP was cloned and sequenced. The primary structure of the protein possessed the PepN-conserved motif GxMEN and the zinc-binding motif HExxHx(18)E, and showed 88% identity with that of PepN from Streptomyces lividans strain 66. We succeeded in overproducing a His-tagged recombinant enzyme using Escherichia coli. The enzyme had a 1.5-fold higher activity in the presence of cobalt ions than in their absence. To evaluate the possible application of TH-4AP to decrease the content of bitter peptides, we investigated the ability of Streptomyces aminopeptidases to hydrolyze synthetic peptides by a coupling method using L-amino acid oxidase and peroxidase. The substrate specificity of TH-4AP toward synthetic peptides was significantly different from that toward aminoacyl-p-nitroanilide derivatives.