The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.