The keratinocyte culture has been used for the reconstruction of artificial skin or as in vitro skin model. For these purpose, keratinocytes should be cultured for long time (usually 10 cumulative population doublings) and it is important to evaluate the state of replicative senescence or cell senescence during this time. This study was undertaken to investigate senescence associated beta-galactosidase (SA-beta-gal) activity for the senescent cell and keratin 19 immunohistochemistry for the skin stem cell in keratinocytes on ten times serial subculture (referred to 10 cumulative population doublings). Keratinocytes were isolated from foreskin of a 2 day old child, and cultured in KSFM and then in DMEM/F-12. In both keratinocytes cultured in KSFM and DMEM/F-12, lysosomal beta-galactosidase activity, which is detected at pH 4.0, was present in all cells regardless of their replicative age. In contrast, SA-beta-gal activity, which is detected at pH 6.0, was present in a few cells (from 0.1% to 3%) during whole subcultures. These data suggest that most of keratinocytes did not undergo replicative senescence in this culture. Furthermore, in keratin 19 skin stem cell staining, a lot of keratinocytes (13.8%) showed strong positive reaction on the 10th subculture. Together with the results of beta-galactosidase activity, the persistence of high proportion of keratin 19 positive skin stem cells implies further increment of keratinocyte populations by continued subcultures.