Desmoyokin, a high-molecular-weight protein of 680 kD with a 170-nm-long dumbbell shape, was originally thought to be localized to the desmosomal attachment plaque and to work as a kind of stabilizer of desmosomes. Recently, desmoyokin was shown to be widely detected in many types of cells that do not possess desmosomes. The purpose of the present study was to elucidate the precise localization and possible function of desmoyokin in human epidermis. In 0.2-micron ultrathin cryosections of human skin for immunofluorescence, anti-desmoyokin antibody showed a ladder-like staining pattern along the cell surface, whereas anti-desmocollin and anti-desmoplakin antibodies as controls showed a discontinuous dotted staining pattern, indicating their distinct localization. Post-embedding immunoelectron microscopy with cryofixation and cryosubstitution revealed that desmoyokin was localized mainly along the non-desmosomal and non-hemidesmosomal plasma membrane, but not to the desmosomes and hemidesmosomes themselves. This localization was further confirmed by double-labeling immunoelectron microscopy with antibodies against desmocollin, desmoplakin, or bullous pemphigoid antigen. Results indicate that desmoyokin was not localized to the desmosomes themselves as previously considered. Desmoyokin was localized to the non-desmosomal and non-hemidesmosomal epidermal keratinocyte cell surface as a plasma membrane-associated protein, and might play a role in cell adhesion that is not directly associated with desmosomes or hemidesmosomes.