The basal layer of the epidermis and hair follicles is composed of actively cycling, transit-amplifying cells and quiescent cells including stem cells. To determine which population is the target of carcinogenic chemicals, we treated CD-1 female mice topically with 5-fluorouracil (5-FU), an agent known to kill cycling but not quiescent cells, to probe the origin of the neoplastic lesions. We first determined that 5-FU kills cycling cells in the epidermis. Treatment of mice at 59 days of age (when in anagen 1) with topical 5-FU delayed hair regrowth by 10 days compared to vehicletreated controls, suggesting that 5-FU killed the cells in anagen. Moreover, 5-FU suppressed the usual hyperplastic response of the epidermal cells to treatment with 12-O-tetradecanoylphorbol-13-acetate. 5-FU reduced the number of epidermal basal cells counted in cross-sections of skin and suppressed DNA synthesis. Approximately 50% of mice treated with 5-FU developed, within 1 week of treatment, a sloughing of the epidermis persisting for 3 weeks, followed by complete healing. Despite the evidence of cell killing in the epidermis and lower hair follicles, in a carcinogenesis experiment where 5-FU or vehicle was applied following tumor initiation with 7,12-dimethylbenz[a]anthracene, the papilloma and carcinoma responses were virtually identical whether or not the mice were treated with 5-FU, suggesting that the tumors arose from quiescent, rather than actively cycling, epidermal cells. When 5-FU was applied before initiation, the papilloma but not the carcinoma responses were slightly but significantly reduced relative to controls. These results are consistent with the hypothesis that the quiescent rather than the rapidly proliferating cells are the targets of tumor initiation.