To better comprehend the role of CHX in the preservation of resin–dentin bonds, this study investigated the substantivity of CHX to human dentin.Dentin disks (n=45) were obtained from the mid-coronal portion of human third molars. One-third of dentin disks were kept mineralized (MD), while the other two-thirds had one of the surfaces partially demineralized with 37% phosphoric acid for 15s (PDD) or they were totally demineralized with 10% phosphoric acid (TDD). Disks of hydroxyapatite (HA) were also prepared. Specimens were treated with: (1) 10μL of distilled water (controls), (2) 10μL of 0.2% chlorhexidine diacetate (0.2% CHX) or (3) 10μL of 2% chlorhexidine diacetate (2% CHX). Then, they were incubated in 1mL of PBS (pH 7.4, 37°C). Substantivity was evaluated as a function of the CHX-applied dose after: 0.5h, 1h, 3h, 6h, 24h, 168h (1 week), 672h (4 weeks) and 1344h (8 weeks) of incubation. CHX concentration in eluates was spectrophotometrically analyzed at 260nm.Significant amounts of CHX remained retained in dentin substrates (MD, PPD or TDD), independent on the CHX-applied dose or time of incubation (p<0.05). High amounts of retained CHX onto HA were observed only for specimens treated with the highest concentration of CHX (2%) (p<0.05).The outstanding substantivity of CHX to dentin and its reported effect on the inhibition of dentinal proteases may explain why CHX can prolong the durability of resin–dentin bonds.