The increasing survival rate of massive burn patients, whose available donor sites for autografting are very limited, results in the emerging development and requirement of artificial skin [1-4]. The objective of this study is to produce cultured autologous keratinocyte grafts and to develop an optimal skin substitute for permanent wound closure. In the development of skin equivalent, human dermal fibroblasts were seeded to form three-dimensional dermal replacement tissue. The keratinocytes were initially cultured in keratinocyte serum free medium supplied with epidermal growth factor (EGF). After two days, the medium was changed to keratinocyte basal medium (without EGF) and subsequently cultured for 14 days by the air-liquid interface culture method. We found that time modulation of EGF has great effect on keratinocyte cell behavior. It is suggested that epidermal keratinocytes with bimedium culture system developed the basement membrane and also differentiated upward in the form of multi-layers.