The basal layer of human epidermis is a heterogeneous population of proliferative and differentiating cells that can be divided into at least three functionally discrete compartments: keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. Basal cells adhere to the underlying basement membrane via integrins, and although decreased adhesion is a key event in epidermal differentiation, the specific role of particular integrins is poorly understood. We report here on the comparative expression and function of the beta1 versus alpha6beta4 integrins in keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells of neonatal human foreskin epidermis. Adhesion assays demonstrate that both keratinocyte stem cells and transit amplifying cells comprise rapidly adhering cells that exhibit high levels of functional beta1 and alpha6beta4 integrins. Interestingly, a proportion of basal cells that have begun to differentiate in vivo within the basal layer as determined by their expression of the differentiation-specific markers K10 and involucrin also retain high levels of activated beta1 integrin, but downregulate alpha6beta4 expression selectively (termed alpha6dimbeta1bri). These cells also retain their adhesive capacity, indicating that induction of differentiation in vivo does not correlate with decreased beta1 integrin expression or function. We have previously reported on the use of alpha6 integrin in conjunction with a proliferation associated marker (10G7 ag) to separate keratinocyte stem cells (phenotype alpha6bri10G7dim) from other basal cells (Li et al. Proc Natl Acad Sci 95:3902-3907 1998). A comparison of the long-term proliferative potential of beta1bri10G7dim cells with alpha6bri10G7dim showed that selection of alpha6bri10G7dim allows the isolation of a purer fraction of keratinocyte stem cells.