Objective To establish a simple and reliable method for isolation and cultivation of epidermal stem cells from neo-natal rat skin basal layer .Methods The single cells were dissociated with twice trypsinization form neonatal rat skin .Thereafter we purified the basal layer stem cells with differential velocity adherent technique with collagen Ⅳ ,and the slow adherent cells were cultured as negative control cells .Both basal layer stem cells and control cells were cultivated with keratinocyte serum-free medium (K-sfm) .Stem cells were identified with β1-integrin and Keratin 19 by co-immunofluorescence assay ,and colony forming assay was executed to evaluate the proliferation potential of stem cells .Results The polygonal cells grew like flagstones ,with doubling time of approximately 24 hours .Both the morphology and growth properties of cells were in accordance with the character of basal layer stem cells .Co-immunofluorescence identification showed the cells were positive for the expression of β1-integrin and Keratin 19 . Basal layer stem cells had stronger clone forming ability in vitro compare with control group .Conclusion The results indicate that two-procedure trypsinization plus differential velocity adhesion is an ideal method for basal layer stem cells separation followed with vigorous vitality and reliable phenotype .