Neuraminidase substrates suitable for analysis of linkage specificity were enzymically synthesized in good yield by linking N-acetylneuraminic acid (Neup5Ac) to O-6 and O-3 of 4-nitrophenyl beta-D-galactopyranoside with beta-D-galactoside-alpha-(2----6)-sialyltransferase and beta-D-galactoside-alpha-(2----3)-sialyltransferase, respectively. By use of these substrates, a convenient colorimetric assay method was developed for the determination of linkage specificity of bacterial and viral neuraminidases. The substrates are incubated with viral or bacterial neuraminidase and subsequently treated with beta-D-galactosidase to convert the liberated 4-nitrophenyl beta-D-galactopyranoside to 4-nitrophenol. The amount of liberated 4-nitrophenol is equivalent to the amount of Neup5Ac released from the substrate, thus allowing measurement of neuraminidase activity. The results showed that bacterial and viral neuraminidases can discriminate between these two compounds, making them useful substrates for the rapid determination of neuraminidase linkage specificity.