Hepatic dysfunction in cystic fibrosis (CF) has been attributed to accumulation of viscous mucoid secretions in intrahepatic bile ducts. The purpose of our study was to compare glycoconjugate secretion by intrahepatic biliary epithelial (IBE) cells derived from normal livers and livers of CF patients with the delta F508 mutation of the cystic fibrosis transmembrane conductance regulator (CFTR). Confluent cells were incubated with 3H-glucosamine (GlcN) for 16 hours, and radiolabeled macromolecules were analyzed for the amount and type of glycoconjugates. Incorporation of 3H-GlcN into macromolecular glycoconjugates was two- to threefold higher in CF cells versus normals, as was uptake of 3H-Glcn into the cytoplasm of CF cells. Gel exclusion chromatography on Sepharose Cl 4B revealed that the secreted glycoconjugates from CF cells eluted entirely in the excluded fraction (molecular weight > 2 × 106), while, in the normal cells, 60% of the glycoconjugates eluted as lower-molecular-weight species. The high-molecular-weight glycoconjugates in both CF and normal cells were identified as chondroitin sulfates, as evidenced by susceptibility to beta elimination, chondroitinase digestion, and amino acid composition. Western blotting of IBE cell secretions with a polyclonal antibody to chondroitin sulfate revealed proteoglycan bands at 100 and 210 kd. Our results indicate that secretion of chondroitin sulfate is markedly increased in CF biliary epithelium in vitro compared with non-CF cells. Increased uptake of precursor 3H-GlcN may contribute to enhanced glycosylation of chondroitin sulfate in CF cells.