The replacement of extensive tracheal defects resulting from intensive care medicine, trauma or large resections is still challenged by the re-epithelialization of an autologous or alloplastic trachea replacement. Therefore, this study was performed to investigate the potential of culture-expanded human respiratory epithelial cells (hREC) to regenerate a functional epithelium for tracheal tissue engineering. hREC from seven male nasal turbinates were freshly isolated, expanded on a collagenous matrix and subsequently cultured in high-density multi-layers to allow epithelial differentiation. The composition of epithelial cells in native respiratory epithelial tissue and culture-expanded hREC was analyzed by histological staining with Alcian blue and by immunohistochemical staining of cytokeratin pairs CK1/10 and CK5/14 with the antibodies 34betaE12 and CD44v6. Differentiation of culture-expanded hREC was further characterized by gene expression analysis of cytokeratins CK5, CK13, CK14 and CK18 using semi-quantitative real-time RT-PCR technique. Histological and immunohistochemical staining of culture-expanded hREC demonstrated basal cells covering the collagenous matrix. These cells formed a cellular multi-layer, which was composed of a basal layer of undifferentiated basal cells and an upper layer of cells differentiating along the squamous metaplasia and ciliated cell lineage. Lineage development of culture-expanded hREC was further documented by the induction of cytokeratins CK13 and CK18. Our results suggest that culture-expanded hREC have the potential to colonize collagen-coated biomaterials and to regenerate epithelial cell types for tracheal tissue engineering.