This study was undertaken to select putative epidermal stem cells from cultured keratinocytes in Cashmere goat fetus and to characterize them in stem cell nature. The keratinocytes were separated enzymatically from fetuses of 12-16 weeks of gestation and co-cultured with mitotically inactivated fetal skin fibroblasts. Putative epidermal stem cells were selected by rapid adherence on collagen type IV substrate and maintained in three different medium conditions: high Ca(2+) concentration, low Ca(2+) concentration, and low Ca(2+) concentration with 50% conditioned medium. The results indicated that epidermal basal cells grew clonally on the feeder layer and were maintained for approximately 48 population doublings without showing signs of replicative senescence. Clonal analysis revealed the presence of three clonal types: holoclones, meroclones and paraclones. Selected keratinocytes on collagen IV substrate could be propagated serially in three medium conditions and the population showed high colony formation efficiency, the same morphology with a high nuclear to cytoplasmic ratio and positive expression of p63, Keratin 19, Keratin 15 and CD71, which are believed to be possible specific markers for keratinocyte stem cells. This study reports a method to isolate a selected keratinocyte population with the characteristics of stem cells.