Organ culture of guinea pig trachea was performed in the presence of [35S]sulfate in order to characterize the sulfated glycoproteins released from the respiratory epithelium and mucosa. The sulfated macromolecules that were synthesized during a 6-h incorporation were separated by CsBr density-gradient centrifugation and gel-filtration chromatography successively. Most of the sulfated secreted macromolecules corresponded to a population of glycoproteins sensitive to reductive beta-elimination but resistant to both chondroitinase ABC and heparinase. These glycoproteins had different buoyant densities (ranging from 1.48 g/ml to 1.16 g/ml) and could be subfractionated according to molecular mass. A major part of the radioactivity was incorporated into high-molecular-mass mucins that were excluded from a Sepharose CL-2B column and did not penetrate into polyacrylamide gel in PAGE. However, a mixture of sulfated O-glycoproteins of much lower molecular mass was also characterized in addition to low amounts of chondroitin sulfate. Epithelial goblet cells are the predominant mucin-containing cells of the respiratory guinea pig trachea. Our results suggest that a wide range of sulfated O-glycoproteins are secreted by the guinea pig tracheal mucosa.