Skin is the largest organ, providing an outer layer of the body and thus creating a barrier against the surrounding environment. It possesses many important functions and can be damaged by various mechanisms. The substitution of damaged skin, mainly in the treatment of deep extensive burns, represents a key challenge. Damaged skin can be replaced permanently by skin autografts, cultured autogenous and allogenic keratinocytes or by the combination of skin substitutes with in vitro cultured autologous keratinocytes. In this article the preparation method and the possible applications of cultured autologous and allogeneic keratinocytes are described; we have long experience of this issue at the Department of Burns and Reconstructive Surgery in the University Hospital Bratislava.