The fucosyltransferase activities of three insect cell lines, MB-0503 (from Mamestra brassicae), BM-N (from Bombyx mori) and Sf-9 (from Spodoptera frugiperda), were investigated and compared with that of honeybee venom glands. Cell extracts and venom gland extracts were incubated with GDP-[14C]fucose and glycopeptides isolated from human IgG and from bovine fibrin. The labeled oligosaccharide products were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, fluorescence marked with 2-aminopyridine and analyzed both by reversed-phase and size-fractionation HPLC. They were identified by their elution positions before and after exoglycosidase treatment in comparison with standard oligosaccharides. These experiments revealed distinct fucosylation potentials in the three cell lines tested. While MB-0503 cells, like honeybee venom glands, are able to transfer fucose into alpha 1-3 and alpha 1-6 linkage to the innermost N-acetylglucosamine, only alpha 1-6-fucosyl linkages were detected with BM-N and Sf-9 cells.