Prolongation of the initial periodate oxidation step of the periodate-borohydride technique/potassium hydroxide/periodic acid-Schiff and periodic acid-Thionin Schiff/potassium hydroxide/periodic acid-Schiff sequences produced little or no change in the diagnostic staining for the potassium hydroxide/periodic acid-Schiff effect, exhibited by the colonic epithelial mucins of man and rat and the Brunner's gland mucin of rabbits. In contrast, there was a gradual, but clear decrease in the intensity of such staining of bovine submaxillary gland mucins. It was concluded that, in the intestinal mucins studied the potassium hydroxide/periodic acid-Schiff effect was due to sialic acids bearing O-acyl substitutents at positions C7 and/or C8 whereas in bovine submaxillary gland mucin the potassium hydroxide/periodic acid-Schiff effect is probably due, at least in part, to the presence of 9-O-acyl sialic acids. This investigation has led to the development of a technique which can be used to identify 9-O-acyl sialic acids.