The sialic acids can have O-acetyl esters at the 4, 7, 8, and 9 positions. Most methods for the detailed study of such molecules require their release from glycosidic linkage, followed by extensive purification. The currently used methods for release and purification of sialic acids allow a reasonable qualitative analysis of the diversity of sialic acids from a given biological source. However, for several reasons, quantitative assessment of the degree and type of O-acetylation is not possible with these methods. Previously known problems include the incomplete and nonrandom release of the different sialic acids by both enzymatic and chemical means, and extensive destruction of the O-acetyl esters (de-O-acetylation) during the release and purification. An additional problem, that migration of O-acetyl groups from the 7 or 8 positions to the 9 position can occur under the conditions of release and purification, particularly when the pH is above 6 or below 3.0, is demonstrated here. It is shown that the O-acetyl esters on free sialic acids are relatively more stable under acid conditions but more labile under basic conditions than similar esters on bound sialic acids. An analysis of the various steps of the conventional purification procedure showed that exposure to the basic anion-exchange resin is the critical step that results in de-O-acetylation and O-acetyl migration. Based upon these and other findings some new methods have been devised, and several modifications of the existing methods have been suggested, that allow the quantitative release and purification of sialic acids with minimal loss of O-acetyl groups. The migration of O-acetyl groups is also decreased by these modifications, but cannot be completely controlled.