We have reported that transglycosylation activity of endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995) J Biol Chem 270: 17723-29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man9GlcNAc, to L-Fuc using Man9GlcNAc2Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2 M L-Fuc was used as acceptor. The transglycosylation produce was purified by high performance liquid chromatography on a graphitized carbon column and the presence of L-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man9GlcNAc-L-Fuc-PA, revealed that a beta-anomeric configuration linkage was formed between GlcNAc and L-Fuc. The GlcNAc was found to be 1,2-linked to L-Fuc by two methods: i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man9GlcNAc-L-Fuc; and ii) preparation of Man9GlcNAc-L-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man9GlcNAc beta (1,2)-L-Fuc. Methyl-beta-L-fucopyranoside, L-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl alpha-L-fucopyranoside, D-Fuc and D-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a 4C1-conformation or equivalent conformation of the acceptor to perform transglycosylation.