Catalytic effects of galactose oxidase on the oxidation of beta-D-galactose-carrying lipids with an oligo-ethylene glycol spacer (number of ethylene glycol units (n) = 1, 2, 3, 6, 9, 13, and 20) were examined. The affinity of galactose oxidase for the galactose residue in the amphiphile (estimated by the inverse of the Michaelis constant, K-m) was much higher than those for free D-galactose and small beta-D-galactopyranosides, and dependent on the length of the ethylene glycol spacer. That is, both below and above the critical micellar concentration, the 1/K-m values decreased with an increase in the n value. The effectiveness of the enzyme, which can be estimated by the k(cat)/K-m value, showed the same tendency as the 1/K-m value. These results could be attributed to the role of the nonpolar environment around the galactose residue in the binding by the enzyme. A significant enhancement of the enzymatic oxidation of galactose residue on the liposome surface was also observed.