H-1 NMR spectroscopy has been used to investigate the mechanism of hydrolysis of the synthetic substrate 2-O-(4-methylcoumarin-7-yl)-5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosidonic acid using the bacterial sialidase from Salmonella typhimurium. Th e H-1 NMR results clearly demonstrate that the initial product of the substrate hydrolysis is the alpha-anomer of N-acetyl-D-neuraminic acid (Neu5Ac). Release of N-acetyl-alpha-D-neuraminic acid as the first product of substrate cleavage is consistent with previous investigations which conclude that both viral and other bacterial sialidases catalyze the hydrolysis of alpha-Neu5Ac ketosides with overall retention of anomeric configuration.