L-Galactose (L-Gal) containing N-glycans and cell wall polysaccharides have been detected in the L-Fuc deficient mur1 mutant of Arabidopsis thaliana. The L-Gal residue is thought to be transferred from GDP-L-Gal, which is a structurally related analog of GDP-L-Fuc, but in vitro L-galactosylation activity has never been detected. In this study, we carried out preparative scale GDP-L-Gal synthesis using recombinant A. thaliana GDP-mannose-3',5'-epimerase. We also demonstrated the L-galactosylation assay of mouse alpha 1,6-fucosyltransferase (MmFUT8) and A. thaliana a1,3-fucosyltransferase (AtFucTA). Both fucosyltransferases showed L-galactosylation activity from GDP-L-Gal to asparagine-linked N-acetyl-beta-D-glucosamine of asialo-agalacto-bi-antennary N-glycan instead of L-fucosylation. In addition, the apparent Km values of MmFUT8 and AtFucTA suggest that L-Fuc was preferentially transferred to N-glycan compared with L-Gal by fucosyltransferases. Our results clearly demonstrate that MmFUT8 and AtFucTA transfer L-Gal residues from GDP-L-Gal and synthesize L-Gal containing N-glycan in vitro. (C) 2016 Elsevier Inc. All rights reserved.