The binding of N, N',N",N" to hen egg white lysozyme was investigated by fluorescence and 1H NMR spectroscopy. From observation of changes in the fluorescence intensity, the association constants of (GlcNAc)4-ol and (GlcNAc) 3 were found to be 0.70 x 105 and 1.07 x 105 M -1, respectively, at pH 5.0 and 30°C. The lack of a substantial difference between the association constants suggests that the binding mode of the (GIcNAc) 3 moiety of (GIcNAc)4-ol is basically similar to that of (GIcNAc) 3, but that the N-acetylglucosaminitol residue of (GIcNAc)4-ol does not interact significantly with lysozyme. On the other hand, 1H NMR spectroscopy revealed a minor difference in the binding modes of the two saccharides. For most of the 1H signals responding to saccharide binding, such as those of Trp 63 H 2, Trp 28 H 5, and lie 98 H vl, the chemical shift changes induced by (GIcNAc)4-ol were almost identical to those induced by (GIcNAc) 3. However, the effect of binding on the signals of Asn 59 H '~ and Trp 108 indole N1H, which are located near subsite C, was different for (GlcNAc)4-ol and (GIcNAc) 3. Thus it is inferred that the binding mode of the first sugar residue of (GlcNAc)4-ol to subsite C is somewhat different from that of (GlcNAc) 3.