It is well documented that the bulge of hair follicle is a 'niche' for a significant population of mouse keratinocyte stem cells, and 95% of rodent clonogenic keratinocytes originate from the bulge region. The ability to form colonies in vitro is a well recognized test for keratinocyte stem cells. We analyzed the epidermis of seven mouse strains and their segregating crosses [(BALB/c x C57BL/6)F1; (BALB/c x CB6F1); (C57BL/ 6 x CB6F1); (CBF1 x CBF1)F2] for their clonogenic activity in vitro. We found that keratinocyte colony (KC) number is a new quantitative multigenic trait. The analysis of KC size in two parental strains (C57BL/6 and BALB/c), the F1 generation and the segregating crosses demonstrated that the size of KC is a quantitative complex trait also. We determined that mouse epidermis has at least two subpopulations of keratinocytes that gave small (< 2 mm2) and large (> 2 mm2) colonies. The differences in the number of small and large colonies between parental strains (C57BL/6, BALB/c) were significant (P < 0.01). A genome-wide scan of the intercross and the two backcrosses maps the number of small KC to the central region of mouse Chromosome 9 (genomewide P value = 0.01). We define this locus as Ksc1. The proximal region of chromosome 4 is associated with the high number of large KC. We defined this locus as Ksc2. We found that Ksc1 and minor loci on chromosomes 6 and 7 map close, if not equal to, loci associated with mouse skin carcinogenesis. We conclude that mouse epidermis has at least two subpopulations of clonogenic keratinocyte stem cells that are regulated by different genes. We suggest that keratinocyte stem cells responsible for small colonies may play a major role in the regulation of resistance or sensitivity to skin carcinogenesis. Investigation of the genes regulating the stem cell number should provide new insight into the mechanisms of skin carcinogenesis, and should help to develop new approaches for therapies not only against active proliferating tumor cells but also quiescent tumor stem cells.