Aminopeptidase H was purified from fresh porcine muscle by ammonium sulfate fractionation and successive chromatographies of DEAE-cellulose, Ultrogel AcA 34, Phenyl-Sepharose CL-4B, hydroxylapatite, and a second DEAE-cellulose. The purified enzyme migrated as a single band on SDS-PAGE. Aminopeptidase H has activity against both L-leucine β-naphthylamide (LeuNap) and α-N-benzoyl-DL-arginine β-naphthylamide (BzArgNap). The molecular weight of the purified enzyme was 51,000 on SDS-PAGE and 390,000 on an Ultrogel AcA 34 column chromatography. The optimum pH for the hydrolysis of LeuNap and BzArgNap was 8.0. The Km values for the hydrolysis of LeuNap and BzArgNap were 0.37 and 1.25mM, respectively. These activities were strongly inhibited by monoiodoacetic acid and leupeptin, but not affected by EDTA, phenylmethylsulfonyl fluoride, pepstatin, or bestatin. The enzyme had the large activities against Met-, Leu-, Lys-, Ala-, Glu-, and SerNap and no activity against ProNap.