Human skin substitutes (HSS) have been developed for repairing burns and other acute or chronic wounds. But although the clinical utility of HSS is well known, scant attention has been paid to their cosmetic properties, especially with regard to color compatibility with the patient's complexion. In this study, we generated an HSS from mixed cell slurries containing keratinocytes and fibroblasts with and without melanocytes on the back of severe combined immunodeficient mice by means of a spontaneous cell-sorting technique. At 16 wk after grafting, Caucasian donor-derived HSS with melanocytes were macroscopically clearly darker than those without melanocytes, and a more darkly pigmented HSS was produced when cells from donors of African descent were seeded. Immunohistochemistry of c-kit, S-100, and HMB45, as well as Fontana-Masson staining and transmission electron microscopy (TEM) demonstrated that melanocytes spontaneously localized to the basal layer. Melanosome transfer to keratinocytes was correctly reorganized, and melanin was evenly dispersed in the basal and suprabasal layers. Colorimetric analysis showed a significantly lower L-value by day 14 following irradiation with 120 mJ per cm2 ultraviolet-B (UVB) (p<0.01), whereas epidermal thickness increased by 50% 1 d after exposure (p<0.01), indicating a normal physiological response to UVB irradiation. These findings suggest that HSS with spontaneously sorted melanocytes offer a means of treating both the structural and cosmetic aspects of skin conditions and trauma, such as pigmentary disorders and skin wounds, by allowing manipulation of the color and population of donor melanocytes.