Based on functional studies in the bone marrow, it has been suggested that the ability to efflux Hoechst 33342 may represent a universal stem cell trait. In this phenotypic and functional characterization of the Hoechst side population (SP) in adult murine epidermis, we demonstrate that these cells are a rare subset of the keratinocyte stem cell-enriched alpha(6)(bri)CD71(dim) fraction comprising SSC(low)/K14(+)/CD34(-)/Oil red O(-)/c-kit(-)/CD45(-) keratinocytes. Epidermal SPs have the smallest cell and nuclear size but exhibit the highest nuclear-to-cytoplasmic ratio of any fraction examined, consistent with a primitive cell type. Although SPs demonstrated poor cumulative in vitro proliferative output, they exhibited sustained epidermal tissue-regenerative activity in vivo compared with unfractionated and non-SP cells. Collectively, these results indicate that the epidermal SP contains the most potent keratinocyte stem cell population in skin epithelium.