Extracellular matrix (ECM) molecules, including those present in basement membranes, are known to play an important role in morphogenesis and differentiation. Primary rat tracheal epithelial (RTE) cells grown on permeable membranes in air-liquid interface (ALI) cultures differentiate into mucous and ciliated cells. We previously showed that RTE cell differentiation is accelerated and enhanced on type I collagen gel-coated membranes. The purpose of this study was to determine whether type I collagen gel matrix also regulates basement membrane formation by RTE cells and whether this correlates with differentiation. Therefore, we examined the synthesis and deposition of several ECM molecules by RTE cells maintained on uncoated and type I collagen gel-coated permeable membranes in ALI cultures. Fibronectin, thrombospondin-1, and alpha-1 type IV collagen gene expression were down-regulated in cultures maintained on type I collagen gel-coated membranes and in differentiated cultures. Fibronectin and laminin alpha, beta, and gamma protein levels also were decreased slightly in response to type I collagen gel and differentiation. Deposition of fibronectin and laminin were dependent on type I collagen gel substratum. While fibronectin and laminin deposition were evident underlying both undifferentiated and differentiated cultures on type I collagen gel, deposition of these proteins on uncoated membranes was not readily detectable in undifferentiated cultures and was limited to patches of fibronectin deposition beneath differentiated cultures. Evidence suggestive of a discontinuous basal lamina-like structure was most apparent underlying differentiated cultures on type I collagen gel. These date demonstrate that ECM RNA and protein expression by RTE cells in ALI cultures are down-regulated by type I collagen gel whereas differentiation and ECM deposition are accelerated and enhanced on type I collagen gel.