We have found that the removal of chondroitin sulfate (CS) GAG chains from dentin matrix significantly decreased bonding to dentin. Objective: To assess the effects of CS removal on collagen matrix organization in dentin. Methods: Bovine dentin fragments were fixed with 10% buffered formalin solution, demineralized with 10% phosphoric acid, rinsed, and embedded in paraffin. Five μm-thick sections were cut and digested with 10μU of chondroitinase ABC (C-ABC) and subjected to immunohistochemistry with monoclonal antibody (2B6) that recognizes the C4S glycosaminoglycan (GAG) moiety. Control sections were incubated in the digestion buffer without C-ABC.  To characterize collagen matrix, 100 μm-thick dentin sections were cut, demineralized with 10% phosphoric acid 1 hr, rinsed, treated with or without C-ABC, and stained with Picrosirius red. In another experiment, C-ABC digested and undigested dentin specimens were observed under SEM. Results: Significant immunoreactivities for C4S were detected in the C-ABC digested group primarily in intratubular dentin and slight immunostaining in intertubular dentin. No staining was observed in the control. Collagen from C-ABC digested dentin exhibited a normal morphology in a wet state. However, after drying, the morphology was completely lost and, in contrast to the control, it could not be recovered by re-wetting. Under SEM, C-ABC digested dentin exhibited an altered morphology as compared with the control. Conclusions: These results indicate that GAG chains in the dentin matrix contribute to the maintenance of collagen matrix architecture, possibly through their high hydrophilic GAG chains. Supported by NIH grant DE10489