Abstract: Short-wave ultraviolet radiation (UVB) is the most carcinogenic part of the ultraviolet spectrum. The target cells of skin cancer are believed to be the bulge stem cells and/or their offspring, the transit-amplifying cells that reside in the epidermis. However, the amount of UVB penetrating epidermis and reaching the bulge cells is very low, which questions if these cells suffer sufficient DNA damage to transform into cancer stem cells. We performed this study to determine whether UV-induced squamous cell carcinoma (SCC) originates from the epidermis or the hair follicles in mice. Hairless mice had their epidermis removed at different levels using CO2 laser ablation. Simulated solar irradiations were administered either preoperatively (in total 7 weeks) or pre- and postoperatively (in total 30 weeks). Control groups were untreated or treated only with solar-simulated radiation or with laser. Blinded clinical assessments of skin tumors were carried out weekly during 12 months observation. Only mice irradiated with solar-simulated radiation both pre- and postoperatively developed tumors. Median time to first, second and third tumor ranged from 19 to 20.5 weeks and was not significantly different between the non-laser and laser-treated groups (P > 0.05). The tumor response was thus similar in UV-exposed mice whether they had their epidermis removed or not. No tumors appeared in control groups. Hence, UV-induced SCC of mice originates from cells of the hair follicle, presumably the bulge stem cells, indicating that ultraviolet radiation penetrates epidermis sufficiently to cause irreversible DNA damage in cells located beneath the epidermis.