Biliary mucin was isolated from human hepatic bile, and its induced effects on the appearance time of cholesterol monohydrate crystals (nucleation time) and on the precipitation of calcium carbonate were studied in vitro to examine the possible significance of mucin for ductular gallstone formation. Mucin was isolated by gel filtration on Sepharose CL-4B and a subsequent CsCl density gradient ultracentrifugation. Mucin thus obtained had a high purity as shown by a high-molecular-weight band on SDS-polyacrylamide gel electrophoresis and by the compatible amino acid composition with mucin purified from the gallbladder. The mucin at as low a concentration as 100 micrograms/ml significantly shortened the cholesterol nucleation time in the supersaturated model bile, mimicking human hepatic bile. On the other hand, the addition of mucin inhibited calcium carbonate precipitation in vitro. Taking account of that both cholesterol and calcium salts are major constituents of ductular gallstones, we conclude that biliary mucin is likely to play an important regulating role in the formation of ductular stones.