A cDNA fragment encoding human salivary-gland kallikrein, including the kallikrein-owned signal peptide, was inserted into a baculovirus vector adjacent to the polyhedrin promoter and expressed in transfected insect cells. Biologically active kallikrein was isolated to homogeneity from serum-free culture supernatant using a four-step protocol. The N-terminal amino acid sequence of the insect-derived kallikrein was identical to that of the natural proteinase, thus indicating the proper removal of the mammalian signal peptide.