Filaggrin and trichohyalin are keratin intermediate filament‐associated proteins, and are primarily expressed in the granular cells of the epidermis and in the inner root sheath cells of the hair follicles, respectively. These two proteins are, however, occasionally co‐expressed in some tissues. To gain more insights into the mechanisms for expression and processing of (pro)filaggrin and trichohyalin during various pathways of epithelial differentiation, we compared their localization by double immunostaining techniques in normal and psoriatic epidermis, tongue filiform papillae and cultured human epidermal keratinocytes. In normal foreskin, trichohyalin immunoreactive cells were observed only occasionally and, when present, they always co‐expressed filaggrin. Trichohyalin expression occurred, however, in filaggrin‐negative cells as well as in filaggrin‐positive cells in the psoriatic epidermis, tongue papillae and cultured keratinocytes. Filaggrin and trichohyalin were induced independently at different times following calcium shift in cultured keratinocytes. Immunoelectron microscopy demonstrated the distinct intracellular distribution of filaggrin and trichohyalin. Some filaggrin granules were observed in the cells where trichohyalin was diffusely distributed. Likewise some trichohyalin granules were found in the cells with diffuse filaggrin labelling. These results suggest the existence of distinct expression/processing mechanisms of filaggrin and trichohyalin in differentiating keratinocytes.