To demonstrate the presence of kallikrein hK2 in the human prostate and seminal plasma, we used mouse monoclonal antibodies (MAb) against a recombinant hK2-fusion protein. Using one of these MAb 9D5, we detected the presence of several major immunoreactive spots of 22 kDa and minor ones of 31 and 55 kDa in prostate cytosol and seminal plasma. After ion exchange and immunoaffinity chromatography of seminal plasma proteins, the 22-kDa immunoreactive proteins were isolated along with 55- and 75-kDa proteins. The NH2-terminal amino acid sequencing permitted identification of fragments of hK2 and protein C inhibitor, respectively, in the 22- ad 55-kDa bands. Furthermore, immunoblotting experiments in one and two-D gels with two different anti-hK2 MAbs and one polyclonal anti-PCI antibody suggested that the major 55- and 75-kDa bands were covalent hK2-PCI complexes containing either the full-length hK2 chain or only its carboxyterminal fragment in the presence of mercaptoethanol. These results demonstrate for the first time the existence of kallikrein hK2 and suggest that PCI may regulate its activity in seminal plasma.