The cultured skin substitute was created through successive cultivation of fibroblasts and keratinocytes that were combined within a collagen matrix. This collagen matrix was composed of a collagen spongy sheet and a collagen gel. The collagen spongy sheet was designed to produce a honeycomb structure having many holes in which all holes through the sheet were filled with collagen gel. This specific structure thereby allows for the nourishment of the cultured keratinocytes on the surface of the matrix when placed on the graft bed. In this study, autologous cultured skin substitute was applied to a 51-year-old man who had sustained a burn injury. Three sheets of the cultured skin substitute (6 × 9.5 cm) were grafted onto the full-thickness excised wound in the right anterior chest wall. One week after grafting most of the matrix disappeared and stratified keratinocytes were seen to have firmly attached to the underlying tissue. Five weeks after grafting a cornified epidermal layer was seen. Ten months after grafting a mature epidermis and a well-differentiated papillary and reticular dermis replacement were observed. The physical properties and color of this grafted area resemble those of normal skin. In the second test case, autologous cultured skin substitute was applied to a 30-year-old man with a scar remaining after tattoo removal. Eight sheets of the cultured skin substitute (10 × 18 cm) were applied on an excised wound (thickness, 0.02–0.025 in.) of both the fore- and upper arms. The histological appearance of a biopsied skin specimen from the grafted area at 3 months after grafting showed a mature epidermis and a well-differentiated reticular dermis replacement. The regenerated skin at 14 months after grafting showed an excellent result.