Proteoglycans (PGs) are a major group of non-collagenous proteins (NCPs) found in dentin matrix, however, at present little is known about their contribution to dentin bonding. Our recent studies have indicated that chondroitin sulfate (CS)-carrying PGs may play a role in dentin bonding. Objectives: To investigate if trypsin treatment removes CSPGs from dentin matrix. Methods: Dentin fragments were prepared from bovine incisors, demineralized with 10% phosphoric acid for 6 hrs, washed, and digested with TPCK-trypsin in a 0.2M NH₄HCO₃ buffer for 48 hrs at 37°C. As a control, the samples were prepared and treated in the same manner but without trypsin. Some of those dentin fragments were stained with 0.05% cupromeronic blue (CMB) and observed under TEM. In another set of experiments, dentin fragments were fixed with 10% buffered formalin solution, demineralized with 10% phosphoric acid, embedded in paraffin. Five μm-thick sections were then cut and subjected to immunohistochemistry with anti-C4S antibody, 2B6.  Results: Under TEM, there was a significant reduction in CMB staining in the trypsin-digested dentin as compared with the control. Immunoreactivity against C4S was observed mainly in intratubular dentin, however, some staining was also observed in intertubular dentin. Although the pattern of staining was similar, there was a decreased immunoreactivity against C4S in the trypsin-digested dentin as compared with the control. Conclusion: These results indicate that the detrimental effects observed on dentin bonding by trypsinization may be due in part to the removal of C4S-GAG chains as a consequence of the cleavages of the C4S-PG core proteins. Supported by NIH grant DE10489.