Purpose: The objective of this controlled randomized split-tooth study was to investigate the effect of dentinal disinfection with a 0.1% chlorhexidine-digluconate solution on the hybridization of 3 dentin bonding agents with dentin. Materials and Methods: Twenty one non-carious molars were randomly divided into 3 groups according to the restoration material used. Two Class-II-restorations in mesial and distal side were performed. One of the two cavities per tooth was washed with chlorhexidine solution (0.1%) for 1 min according to a random list. Restorations were carried out with one of the three materials: AquaPrime/Resolcin, Optibond/Herculite or Prime&Bond NT/Esthet-X. After sectioning and surface etching (15 s H3PO4 and 120 s NaOCl) the specimens were investigated under SEM. Hybrid layer thickness and tag lengths were documented. Results: Hybrid layers and tags were found in every group. The hybrid layer thicknesses were: It is concluded that using chlorhexidine before acid etching significantly effects hybridization of bonding resins with dentin. Keywords: dentin adhesion, hybrid layer, chlorhexidine