Transglycosylation activity of endo-beta-N-acetylglucosaminidase HS (Endo HS) was investigated using native human transferrin as a donor of an asparagine-linked oligosaccharide and p-nitrophenyl-beta-d-glucose (PNP-beta-d-Glc) as an acceptor of the oligosaccharide. The amount of the product increased dependent on the concentration of the acceptors. Absorption spectrum, exoglycosidase digestion and matrix assisted laser desorption and ionization-time of flight (MALDI-TOF) mass analysis of the transglycosylation product indicated that the asialobiantennary complex type oligosaccharide of human transferrin was transferred to PNP-beta-d-Glc. Endo HS also transferred the oligosaccharide of human transferrin to PNP-alpha-d-Glc, PNP-alpha-d-Gal, PNP-beta-d-Gal, PNP-beta-d-Man, PNP-beta-d-Xyl, PNP-beta-d-GlcNAc, and PNP-glycerol at a different rate. No apparent difference in the K(m) value for human transferrin as an oligosaccharide donor was observed using different acceptors, PNP-beta-d-Glc and PNP-glycerol. The amount of the transglycosylation product successively increased and became constant and then very slightly decreased during the course of enzyme reaction. Endo HS was also transferred the triantennary complex type oligosaccharide of calf fetuin and the bi-, tri-, and tetrantennary complex type oligosaccharides of human alpha(1)-acid glycoprotein to PNP-beta-d-Glc. Furthermore, Endo HS transferred an asparagine-linked oligosaccharide from a hen egg glycopeptide to PNP-beta-d-Glc. The results demonstrate that Endo HS can transfer a wide variety of asparagine-linked complex type oligosaccharides to various monosaccharides. Endo HS was distinct from other enzymes in the specificity for oligosaccharide donors and acceptors.