Although chondroitin sulfate (CS) is known to act as an inhibitory axon guidance cue, retinal axons show substantial growth on a culture substrate containing CS. Thus, the question arises as to how retinal axons elongate on CS-containing culture substrates. To elucidate the effects of retinal axons on a substrate containing CS, we synthesized biotinylated CS (biotin-CS) and developed a culture substrate with streptavidin-conjugated biotin-CS (complex between streptavidin and biotin-CS) to culture retinal axons. The effects of retinal axons on the streptavidin-biotin-CS complex were analyzed immunocytochemically using antibodies against CS and streptavidin, which recognize the carbohydrate and protein portions of the complex, respectively. After the axons were cultured on the substrate, areas that were CS-immunonegative but streptavidin-immunopositive were observed on the surface, corresponding to areas with or without axons, respectively. Absence of CS immunostaining was considered to be caused by structural alterations in the carbohydrate chains of the CS under the influence of the axons.