Abstract: A technique for the culture of outer root sheath keratinocytes from plucked human hair follicles has been developed. The anagen-phase human hairs were plated on 3T3 cell feeder layers and were cultured in DAhEM, substituted with different' growth factors in a CO₂ incubator at 37℃. After 14 days in primary culture, 3T3 cells were removed with 0.02% EDTA and the keratinocytes were detached and disaggregated to single cells by treatment with 0.25% trypsin and 0.02% EDTA at 37℃ for 15 min. The dispersed cells were then replated on fresh 3T3 cell feeder layers. The keratinocytes were serially sub-cultured for 3-4 times. By this technique, large amounts of stratified multilayerd kera-tinocyte cultures were readily obtained in primary culture as well as in subcultures. The technique described here is a simple and effestive culture method and provides a large amount of hair follicle keratinocytes for medical research programmes.