N-glycans were released from the SARS coronavirus (SARS-CoV) spike glycoprotein produced in Vero E6 cells and their structures were determined by a combination of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, negative ion electrospray collision-induced dissociation time-of-flight mass spectrometry and normal-phase high-performance liquid chromatography with exoglycosidase digestion. Major glycans were high-mannose (Man(5-9)GlcNAc(2)), hybrid and bi-, tri- and tetra-antennary complex with and without bisecting GlcNAc and core fucose. Complex glycans with fewer than the full complement of galactose residues were present and sialylation was negligible. Treatment with the glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) inhibited N-glycan processing as evidenced by the appearance of glycans of composition Glc(3)Man(7-9)GlcNAc(2). However, some complex glycans remained suggesting the presence of an alpha-endomannosidase. Our data in tissue culture indicate that inhibition of N-glycan processing may be considered as a therapeutic strategy against SARS CoV infections.N-glycans were released from the SARS coronavirus (SARS-CoV) spike glycoprotein produced in Vero E6 cells and their structures were determined by a combination of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, negative ion electrospray collision-induced dissociation time-of-flight mass spectrometry and normal-phase high-performance liquid chromatography with exoglycosidase digestion. Major glycans were high-mannose (Man(5-9)GlcNAc(2)), hybrid and bi-, tri- and tetra-antennary complex with and without bisecting GlcNAc and core fucose. Complex glycans with fewer than the full complement of galactose residues were present and sialylation was negligible. Treatment with the glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) inhibited N-glycan processing as evidenced by the appearance of glycans of composition Glc(3)Man(7-9)GlcNAc(2). However, some complex glycans remained suggesting the presence of an alpha-endomannosidase. Our data in tissue culture indicate that inhibition of N-glycan processing may be considered as a therapeutic strategy against SARS CoV infections.