The overall aim of this study was to further characterize the activity from three human kallikreins in stratum corneum, among them human kallikrein 14 (hK14), a kallikrein previously not known to exist in epidermis. In our group, two epidermal serine proteases, hK5 (SCTE) and hK7 (SCCE), have been cloned and charachterized. They are believed to be involved in stratum corneum turnover through a proteolytic cascade ending in the breakdown of desmosomal proteins. It was found that a major part of the trypsin-like activity, measured by cleavage of chromogenic peptide substrates, in extracts from plantar stratum corneum, could not be ascribed to hK5. In search for yet another serine protease we have now purified hK14 from human plantar stratum corneum through reversed phase chromatography. A panel of nine individuals were subjected to tape stripping of superficial non palmo-plantar stratum corneum. Protein contents of tape strips were extracted. Western blot of extracts revealed the presence of hK14 in all individuals tested. Quantification experiments of the relative amounts of hK5, hK7 and hK14 in human epidermis showed that the enzymes are present in a ratio of approximately 3:10:1. a1-antitrypsin inhibitor analysis showed that the major part of hK14 in human stratum corneum is present in active form. We have also noted that active hK5, hK7 and hK14 are present in sweat. This finding might have relevance for patients suffering from atopic dermatitis as these often experience a more pronounced itch when sweating. We hereby conclude that hK14 is present in human stratum corneum. hK14 is a major contributor to the trypsin-like activity in human stratum corneum, indicating that hK14, in addition to hK5 and hK7, is likely to have an important function in the physiology of human stratum corneum.