Aqueous solutions of N-acetyl-neuraminic acid (Neu5Ac, 1) labeled with C-13 at C1, C2, and/or C3 were analyzed by C-13 NMR spectroscopy to detect and quantitfy the acyclic forms (keto, keto hydrate, enol) present at varying pHs. In addition to pyranoses, solutions contained the keto form, based on the detection of C2 signals at similar to 198 ppm (similar to 0.7% at pH 2). Spectra of [2-C-13] and (3-C-13] isotopomers contained signals arising from labeled carbons at similar to 143 and similar to 120 ppm, respectively, which were attributed to enol forms. Solution studies of [1,2,3-C-13(3)]1 substantiated the presence of enol (similar to 0.5% at pH 2). Enol was not detected at pH > 6.0. A C2 signal observed at similar to 94 ppm was identified as C2 of the keto hydrate (similar to 1.9% at pH 2), based partly on its abundance as a function of solution pH. Density functional theory (DFT) calculations were used to study the effect of enol and hydrate structure on J(CH) and J(CC) values involving C2 and C3 of these forms. Solvated DFT calculations showed that (2)J(C2,H3) in cis and trans enols have similar magnitudes but opposite signs, making this J-coupling potentially useful to distinguish enol configurations. Solvent deuterium exchange studies of 1 showed rapid incorporation of H-2 from (H2O)-H-2 at H3(axial) in the pyranoses at p(2)H 8.0, followed by slower exchange at H3(equatorial). The acyclic keto form, which presumably participates in this reaction, must assume a pseudo-cyclic conformation in solution in order to account for the exchange selectivity. Weak C-13 signals arising from labeled species were also observed consistently and reproducibly in aqueous solutions of C-13-labeled 1, possibly arising from products of lactonization or intermolecular esterification.