To obtain basic information about the structural diversity and functional specificity of chondroitin sulfates (CSs) in the formation of the retinotectal pathway in chick embryos, the distribution of CSs around the optic tract was investigated by using anti-CS monoclonal antibodies with different specificities. The CSs are unbranched polymers composed of repeating disaccharide units of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). The disaccharide units are classified into O-, A-, C-, D-, and E-units based on the position(s) of the added sulfate group(s). The MO-225 monoclonal antibody recognizes CSs that are rich in the D-unit [GlcA(2S)beta1-3GalNAc(6S)]; the MO-225 epitopes were distributed in the diencephalotelencephalic boundary and the neuropil encircling the optic tract. In addition, they were distributed on membrane surfaces of the retinal axons running in an interface layer in contact with the neuropil encircling the optic tract. The results suggest that D-unit-rich CSs are involved in delimiting the border of the optic tract and in the chronological sorting of the retinal axons.