Epidermis was obtained in vitro after air exposure of keratinocyte cultures grown on a dermal equivalent. Some cultures were established from enzymatically dissociated keratinocytes of either interfollicular epidermis or hair follicle outer root sheath. Others resulted from centrifugal outgrowth of epidermal sheet, out of skin biopsies or hair follicles, which were directly implanted into dermal equivalents. Whatever the system used, a multilayered epidermis was obtained with an overall architecture resembling that of human epidermis. However, depending on the tissue culture method used and the source of keratinocytes, significant differences were observed. The most striking finding was the difference in 67 kDa keratin expression: the only case where it was strictly suprabasal and homogeneously expressed in the cytoplasm of the cells, as in normal epidermis, was found in the epidermis obtained from hair follicle explants. With the other methods, the expression of this marker was delayed and patchy. These results are discussed in terms of possible intrinsic differences between interfollicular and follicular keratinocytes.