In this report several NeuAc analogues differently modified at position C-9 were tested as substrates for CMP sialic acid synthase from bovine brain: the hydroxy group at C-9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group. The synthase was partially purified by chromatography on CDP-hexanolamine--Sepharose. CMP-glycosides synthesized were measured by analytical HPLC at 275 nm. Each NeuAc analogue was activated to the respective CMP-glycoside: Km-values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C-9. CMP-glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500-MHz 1H-NMR data of CMP-9-amino-NeuAc and CMP-9-acetamido-NeuAc were obtained. Each CMP-activated NeuAc analogue was a suitable donor substrate for Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non-labelled NeuAc analogues can be incorporated into glycoprotein with high yield.