Introduction: Reconstruction of large defects of the floor of the mouth after tumour ablative surgery is typically performed using free skin flaps. Drawbacks include donor site defects and hairiness of the flaps. Cultured mucosal substitutes (CMS) have been developed and were clinically used as alternatives. A-cellular dermis is often used as a dermal carrier, as it shows less contraction in vivo than e.g. a collagen gel and is easy to handle by the surgeon. One of the problems in CMS using a-cellular dermal carriers is the varying quality of the epidermis. We investigated the role of fibroblasts on epidermal morphology and proliferation. An in vitro model was used combining cultured mucosal keratinocytes and a-cellular dermis. We present a simple and reliable technique to seed the fibroblasts into the a-cellular dermis using centrifugal forces. Materials and methods: Keratinocytes and fibroblasts were isolated from human buccal mucosa. Fibroblasts were centrifuged into the dermis and keratinocytes were seeded on the papillary side. One and two-week-old CMS were stained for differentiation markers beta-1 integrin, cytokeratin 10 and involucrin, as well as for basal membrane (BM) components, such as laminin 5 and collagen type IV and VII. Furthermore, proliferation was investigated using Ki-67 as a marker. The distribution of the fibroblasts was visualised by vimentin staining. Results: Histology showed a striking and significant increase in amount of basal cells per surface graft, as well an increase in cell-layers of basal keratinocytes as confirmed by b1 integrin staining. In addition fibroblasts enhance the early deposition of BM molecules. Ki-67 staining showed a significant increase in cell proliferation. Finally, we observed that epidermis of fibroblast-containing CMS mature faster into a well organised epithelium than the ones without fibroblast. Conclusion: The authors conclude that the addition of fibroblasts results in a dramatic increase in the quality of the CMS. The higher number of basal keratinocytes in the epidermis resulted in enhanced proliferation in the epidermis. The earlier deposition of BM molecules and early maturation of the epidermis should result in reduced culture times of CMS before transplantation. Also it might lead to advantageous effects on in vivo graft survival.