Although lung epithelial cells directly attach to the basement membrane underneath in vivo, harvested epithelial cells are typically cultured on type I collagen gel (Col I-gel) in vitro. Recently we developed new culture substratum, designated as "synthesized Basement Membrane" (sBM), that has bared lamina densa on fibrillar collagen. To validate the usefulness of sBM substratum in airway tissue reconstitution in vitro, we cultured rat tracheal epithelial cells on sBM substratum and Col I-gel. When starting the air-liquid interface culture, most of the epithelial cells were squamous and positive for the basal cell marker cytokeratin 14 (CK14). After 14 days on sBM substratum, CK14-positive cells differentiated not only to Clara and mucous cells, but also to ciliated cells. Those differentiated cells formed pseudostratified-like epithelium and the remaining CK14-positive cells were polarized to the basal side. However, on Col I-gel, the CK14-positive cells were still squamous and not polarized, and ciliated cells did not appear. In conclusion, we established a new culture model on sBM substratum in which basal cells could differentiate to ciliated cells. The application of sBM substratum is useful in the study of the airway epithelial cell differentiation in vitro.