Several converging lines of evidence suggest that keratinocyte stem cells including those clonogenic in vitro, are the target cells in skin carcinogenesis. However, the mechanism of their involvement in carcinogenesis continues to be unknown. Recently, we have demonstrated that the number of keratinocyte clonogenic stem cells from mice is a genetically defined and quantitative complex trait. In this study, we analyzed the size of the colonies in BALB/c, C57BL/6, and their (BALB/c x C57BL/6)F1 hybrid mice. We found that the size of the colonies is also genetically regulated and that there are at least two types of clonogenic keratinocytes that form either small or large colonies. Small and large colony numbers were significantly different in BALB/c and C57BL/6 mice. We performed a genome-wide scan of small and large colony number in the two backcrosses [BALB/c x (BALB/c x C57BL/6)F1]; [C57BL/ 6 x (BALB/c x C57BL/6) F1]; and [(BALB/c x C57BL/ 6)F1 x (BALB/c x C57BL/6)F1] intercross mice. We report here that the loci on chromosomes 4 and 9 play a role in the regulation of large and small colony number respectively. We conclude that mouse epidermis has two major types of stem cells generating small and large colonies, and regulated by different genes. Our results suggest that the loci, including one major and several minor loci linked to the subpopulation of keratinocytes forming small colonies were related, if not identical to, the loci linked to susceptibility or resistance to skin tumor promotion and skin tumor development.