Synthesized glycolipids have been reported as hydrolase models, and their active sites could be explained on the basis of 2, 3-hydroxyl groups of the sugar moiety. In this study, a glycolipid was found to exhibit acidic nature in water, reflecting the dissociation of the hydroxyl group, and the conjugated base to be concerned with the hydrolysing activity. The 2- or 3-deoxy-glycolipids then were synthesized and used in the hydrolysis of phenylalanine p-nitrophenyl ester. 3-Deoxy-glycolipid showed only a little lower hydrolysing activity than the perfect glycolipid, while 2-deoxy-glycolipid, considerably lower activity. The hydroxyl group on 2-position is thus shown to be a predominant active site, although 3-hydroxyl one also is a weaker active site. That is, the oxy anion on 2-position of a sugar moiety is esti-mated as active species that attack the carbonyl carbon of an ester nucleophilically with consequent bond cleavage.