The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.