The field of hair follicle regeneration is advancing rapidly, and there have been a number of major achievements over the last decade. Nonetheless, most of the current technologies are still unable to maintain their in vivo characteristics in vitro. The formation of new hair follicles for the treatment of alopecia using tissue engineering technology is promising; however, little or no work has been attempted in this area so far. In order to enhance the hair inducing ability of epithelial cells and to form the functional epithelial structure, we attempted to fabricate threedimensional scaffolds by using bladder submucosal membrane (BSM), keratin, hyaluronic acid (HA), and mouse newborn epithelial skin scaffold (NESS). We characterized their properties with respect to the formation of the epithelial structure. The BSM sponge demonstrated the intrinsic activation of melanocytes once we inserted the inductive dermal papilla sphere. We found that the fiber sheet made of keratin enhanced cell spreading and adhesion better than the sheet with HA. The human outer root sheath keratinocytes formed a cluster on the NESS. These scaffolds would be used for the follicular epithelial cell physiology and study for property of them in vitro. This study demonstrated the potential of epithelial cell scaffolds for effective hair regeneration.