Formalin-fixed tissue specimens containing glycoproteins with side chain O-acylated sialic acids were used to re-examine, compare and evaluate the usefulness of three methods based on the periodic acid-borohydride reduction-saponification-periodic acid-Schiff sequence (PA-Bh-KOH-PAS) for the histochemical identification of 9-O-acyl sialic acids (9-O-AcSA). Method I, modified from Veh et al. (1979), involved a comparison of the staining intensely obtained when both oxidation steps of the PA-Bh-KOH-PAS sequence were carried out with the selective oxidation technique of Volz et al. (1987) with that obtained when the initial oxidation step was carried out with 0.5 M periodic acid for 4 h at room temperature. Methods II and III, modified from Reid et al. (1978), involved an initial PA-Bh step under oxidation conditions that cleaved all the vicinal diols associated with neutral sugars and side chain unsubstituted and 7-O-acyl sialic acids. The Schiff staining obtained following subsequent re-oxidation with either 0.5M (method II) or 1% periodic acid (method III) for 4 h at room temperature (PA-Bh-PAS procedure) identifies 9-O-AcSa.(ABSTRACT TRUNCATED AT 250 WORDS)