Per-O-methylated cyclodextrins containing a single 2-O-(2-acetate), 2-O-(3-propanoate) or a 6-carboxylate were investigated for glycosidase activity on p-nitrophenyl glycosides. The former two compounds displayed enzyme catalysis giving rate accelerations of 500-1000, while the latter compound gave marginal catalysis. These results show that per-O-methylated cyclodextrins direct substrate binding from the secondary face leading to better catalysis.