The target site for N-linked biantennary and triantennary oligosaccharides containing multiple terminal Le(x) determinants was analyzed in mice. N-linked oligosaccharides containing a single tert-butoxycarbonyl-tyrosine attached to the reducing end were used as synthons for human milk alpha-3/4-fucosyltransferase to prepare multivalent Le(x) (Gal beta 1-4[Fuc alpha 1-3]GlcNAc) terminated tyrosinamide oligosaccharides. The oligosaccharides were radioiodinated and examined for their pharmacokinetics and biodistribution in mice. The liver was the major target site in mice at 30 min, which accumulated 18% of the dose for Le(x) biantennary compared with 6% for a nonfucosylated Gal biantennary. By comparison, Le(x)- and Gal-terminated triantennary accumulated in the liver with a targeting efficiency of 66 and 59%, respectively. The liver targeting of Le(x)-biantennary was partially blocked by co-administration with either galactose or L-fucose whereas Le(x) triantennary targeting was only reduced by co-administration with galactose. In contrast to these results in mice, in vivo experiments performed in rats established that both Le(x) and Gal terminated biantennary target the liver with nearly identical efficiency (6-7%). It is concluded that the asialoglycoprotein receptor in mice preferentially recognize Le(x) biantennary over Gal biantennary, whereas little or no differentiation exists in rats. Thereby, the mouse asialoglycoprotein receptor apparently possesses additional binding pockets that accommodate a fucose residue when presented as Le(x).