To explore the expression and gain more information on the function of transglutaminase 5 enzyme in normal and defective human epidermis, we generated a rat antihuman transglutaminase 5 antiserum elicited against a purified active recombinant protein expressed in the baculovirus system. By use of Western blotting and immunofluorescence methods, the immunospecificity of the antibodies for transglutaminase 5 was tested; no crossreactivity with other transglutaminases (types 1, 2, and 3) was observed, thus allowing histochemistry studies. By indirect immunofluorescence analysis the antibodies decorated the upper layers of normal human epidermis, with consistent staining in the spinous and granular layers. We evaluated transglutaminase 5 expression in comparison with proliferating (keratin 14) and differentiating (transglutaminase 3) markers in different diseases, such as psoriasis, ichthyosis vulgaris, lamellar ichthyosis, and Darier's disease. We observed that transglutaminase 5 contributes, as a secondary effect, to the hyperkeratotic phenotype in ichthyosis (both vulgaris and lamellar) and in psoriasis. In Darier's disease, transglutaminase 5 expression, as well as transglutaminase 3, is completely missregulated, being overexpressed or totally absent in different areas of the same lesion.