Human granzyme 3 (Gr3) is a serine protease contained in the granules of natural killer cells and cytotoxic T lymphocytes. To elucidate the biochemical and physiological characteristics of Gr3, we attempted to prepare an enzymatically active recombinant human Gr3 without refolding and proteolytic activation. An expression vector was constructed, in which the pre-/pro-peptide coding sequence of Gr3 was replaced with the bacterial pelB leader sequence. The resultant expression product was a fully active protease in the periplasmic fraction of Escherichia coli and was purified to homogeneity. The purified enzyme effectively hydrolyzed Z-Lys-SBzl, a conventionally used substrate of Gr3. In addition, it also hydrolyzed the peptide substrate library FRETS-25Xaa series, required basic amino acid residues, Arg or Lys, at the P1 position, and most efficiently hydrolyzed the carboxylic side of Phe-Tyr-Arg downward arrow (P3-P2-P1) sequence of the 475 tripeptide combinations.