We and others have shown previously that corneal keratinocyte stem cells can proliferate in vitro better than their progeny cells. In this paper, we applied this approach to the identification of hair follicular stem cells. When human scalp hair follicles were placed in explant culture, the bulge area yielded best outgrowths. In another experiment, we isolated different subpopulations of human follicular keratinocytes by micro-dissection, dispersed them by trypsin/EDTA into single cells, and grew them in the presence of 3T3 feeder cells. The keratinocytes were then subcultured under identical conditions to compare their in vitro life span. Our results indicate that the life span of keratinocytes of the upper follicle (containing mainly the isthmus area) > sebaceous gland > lower follicle (between the bulge and bulb) > bulb (containing the matrix cells). The cultured upper follicular keratinocytes tend to be small and relatively uniform in size. The poor in vitro growth of matrix cells may reflect their non-stem cell nature and/or special growth requirement(s) satisfied in vivo by the neighboring dermal papilla cells. Unexpectedly, we found that the upper follicular keratinocytes grow even better than epidermal keratinocytes. The existence of a subpopulation of keratinocytes with an in vitro growth potential superior than other known keratinocytes of the skin supports the hypothesis that follicular stem cells reside in the upper follicle. Our data also raise the possibility that putative follicular stem cells are involved not only in forming the follicle, but also in the long-term maintenance of the epidermis. Finally, we discuss the possibility that keratinocyte stem cells, as defined by their in vivo slow-cycling nature, are absent in culture.