Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins which recognize a fucose residue were observed over a wide range of molecular weights, compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping HPLC and MALDI-TOF MS confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin (AAT), alpha1-acid glycoprotein (AGP) and haptoglobin which are synthesized in the liver showed higher fucosylation in bile than in serum. To know a direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8) deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of AAT and AGP were quite low in bile of Fut8 deficient mice as compared to wild type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8 deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.