The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin gel from plasma cryoprecipitate containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on the gel at different seeding ratios. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained within 10-15 days in culture. Early expression of basal membrane proteins was detected by immunostaining with laminin and type IV collagen antibodies. Cell proliferation was detected both in the epidermal layer and in the fibroblast embedded in the gel as assessed by BrdU incorporation. Detachment of composite cultures from dishes or flasks is a simple and quick procedure without the need for dispase treatment. Grafting of composite cultures to nude mice gave rise to an orderly stratified, orthokeratinized epithelium resembling human epidermis. A number of advantages including a large expansion factor without the need of 3T3 feeder layer, the availability of fibrin/plasma cryoprecipitate from blood banks and the versatile manipulation of composite cultures suggest that this system could be suitable for the definitive coverage of severely burned patients.