Metallothionein (MT) is a low-molecular weight metal-binding protein. Although the physiologic function of MT is not fully known, it is present in various species and various organs including the skin. MT is strongly stained in hyperplastic epidermal tissues in normal skin and in hyperplastic skin lesions, and increased expression of mRNA of the MT gene has been demonstrated in skin stimulated by proliferative agents, suggesting that MT is involved in the proliferation of epidermal keratinocytes. To improve our understanding of the role of MT in epidermal hyperplasia, mice with null mutations in their MT-1 and MT-2 genes were used in this study. We compared the epidermal hyperplasia in MT-null mice and in normal C57BL/6 J mice after treatments with cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B irradiation, which stimulate epidermal proliferation. Immunostaining of MT was not detected in the skin of MT-null mice, and these mice developed significantly less epidermal hyperplasia than the normal mice after exposure to each stimulator. We determined the metal contents of skin samples by the proton-induced x-ray emission method. The zinc content of the skin of the MT-null mice was lower than that of the control mice before stimulation. After stimulation of epidermal hyperplasia, MT-null and normal mice showed significantly reduced levels of zinc. These findings indicate that cellular MT is involved in the proliferative process of the epidermis induced by cholera toxin, 12-0-tetradecanoylphorbol-13-acetate, and ultraviolet B light through its regulatory action on the metal metabolism required for cell growth.