An air-liquid interface (biphasic) primary culture system in which guinea pig tracheal epithelial cells maintain morphologic characteristics of differentiated epithelium has been developed in this laboratory. In this report, we compared quantitatively cell populations of 8-day cultures to those of epithelial mucosa in intact trachea. In addition, high molecular weight glycoconjugates released by the cultured cells were isolated and characterized. Quantitative morphometric analysis revealed similar volume densities of ciliated, secretory, basal, and "other" cells in cultures and in intact tracheal surface epithelium, although the cultures tended to have smaller cells and contained fewer basal cells. High molecular weight glycoconjugates released apically by cell cultures and excluded from Sepharose CL-4B columns contained approximately 5% hyaluronic acid but undetectable amounts of other proteoglycans, such as chondroitin sulfate, heparan sulfate, and dermatan sulfate. The hyaluronidase-resistant glycoconjugates exhibited a peak buoyant density at 1.49 g/ml on cesium chloride density gradient centrifugation and were shown to contain mucin-type carbohydrate to peptide linkages (i.e., GalNAc to ser/thr) and an amino acid composition typical of respiratory mucins. The results indicate that this organotypic cell culture system mimics quite closely morphology of mucosal epithelium in intact airways and that the cells release high molecular weight glycoconjugates with biochemical properties of mucin-type glycoproteins. Thus, this in vitro system appears well-suited for studies of mucin secretion and other functions of respiratory epithelial cells.