Ornithine decarboxylase (ODC) plays a key role in the biosynthesis of polyamines, which are necessary for cell growth and differentiation. ODC expression is very tightly controlled in all normal cells; however, regulation of its expression is altered in many tumor cells resulting in much higher basal levels of ODC in tumors. To investigate the potential role of ODC overexpression in epidermal tumorigenesis, we constructed a replication-defective retroviral vector to overexpress a truncated ODC protein in epidermal cells. Stable viral infection of mouse epidermal cells dramatically increases not only the basal ODC activity but also the basal putrescine and spermidine levels. In all infected epidermal cells with high polyamine levels, DNA synthesis is increased as measured by [³H]thymidine incorporation into DNA as well as increased bromodeoxyuridine staining in the nuclei of ODC-infected epidermal cells. ODC viral infection of nontumorigenic BK-1 epidermal cells and primary cultures of mouse keratinocytes and fibroblasts from newborn mouse skin yields no tumors when injected s.c. into athymic nude mice or when transplanted as skin grafts onto nude mice. Epidermal cell lines SP-1 and 308 (which possess an activated ras^Ha gene) are not tumorigenic when injected s.c. into nude mice. However, following infection with the ODC virus, they form tumors filled with keratin and papilloma-like projections of hyperplastic epidermal cells displaying dysplasia and many mitotic figures. These data indicate that ODC overexpression by itself is not sufficient to induce tumors in normal cells but that increased expression of ODC enhances tumor development in initiated premalignant epidermal cells.