Patients with large burn wounds have a limited amount of healthy donor skin. An alternative for the autologous skin graft is transplantation with autologous keratinocytes. Conventionally, the keratinocytes are cultured with mouse feeder layer cells in medium containing fetal calf serum (FCS) to obtain sufficient numbers of cells. These xenobiotic materials can be a potential risk for the patient. The aim of the present study was to investigate if keratinocytes could be expanded in culture without the need of a feeder layer and FCS. Keratinocytes were cultured on tissue culture plastic with or without collagen type IV coating in medium containing Ultroser G (serum substitute) and keratinocyte growth factor (KGF). An in vitro skin equivalent model was used to examine the capacity of these cells to form an epidermis. Keratinocytes in different passages (P2, P4, and P6) and freshly isolated cells were studied. Keratinocytes grown on collagen type IV were able to form an epidermis at higher passage numbers than cells grown in the absence of collagen type IV (P4 and P2, respectively). In both cases the reconstructed epidermis showed an increased expression of Ki-67, SKALP, involucrin, and keratin 17 compared to normal skin. Only 50,000 keratinocytes grown on collagen type IV in P4 were needed to form 1 cm2 epidermis, whereas 150,000 of freshly isolated keratinocytes were necessary. Using this culture technique sufficient numbers of keratinocytes, isolated from 1 cm2 skin, were obtained to cover 400 cm2 of wound surface in 2 weeks. The results show that keratinocytes can be cultured without the need of a fibroblast feeder layer and FCS and that these cells are still able to create a fully differentiated epidermis. This culture technique can be a valuable tool for the treatment of burn wounds and further development of tissue engineered skin.