To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities. Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-beta-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline's stability and Endo-CC's transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 mu g GlcNAc-RNase B, 200 mu g SG-oxazoline and 3 mu g Endo-CCN180H in 20 mu l 20 mM Tris/HCl pH 7.5 at 30 A degrees C for 30-60 min. This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.