Hamster tracheal epithelial cells growing on type I collagen gel synthesize and secrete high molecular weight glycoconjugates which elute in the void volume upon Sepharose CL-4B column chromatography. The presence of any proteoglycans in this void volume material was ruled out based on both enzymatic analysis and behavior on DEAE-ion exchange chromatography. Based on the incorporation of radioactive precursors, followed by strong acid hydrolysis or neuraminidase digestion, the material was shown to contain sialic acid, fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and sulfate. Complete susceptibility to papain digestion and reductive beta-elimination suggests that the material consists of O-linked glycoproteins. The identification of N-acetylgalactosaminitol in the beta-eliminated oligosaccharides confirms this notion. The molecular weight of the oligosaccharides following beta-elimination ranges from 4,000 to 15,000. We conclude that the high molecular weight glyconjugates produced by hamster tracheal epithelial cells in primary culture are mucous glycoproteins based on size, sensitivity to alkaline borohydride treatment, and monosaccharide composition. Further characterization of these mucous glycoproteins showed both size and charge microheterogeneity among molecules. Detailed structural analysis of oligosaccharides of these mucous glycoproteins is currently under way.