Purpose: This in vitro study evaluated the amount and distribution of outward fluid flow that occurred when an experimental etch-and-rinse hydrophobic adhesive was applied to ethanol-saturated dentin before and after oxalate pretreatment. Materials and Methods: Measurements of dentin permeability were performed under a constant pulpal pressure of 20 cm H2O in deep and middle dentin. A lucifer yellow solution was placed in the pulp chamber to determine the distribution of the water contamination of the hybrid layers. Results: The distribution of fluorescence in dentin specimens that were not pretreated with oxalate revealed that the dye permeated around the resin tags and filled the hybrid layer. Dentin specimens pretreated with oxalate prior to resin bonding, showed 80% to 83% less (p 0.05) water contamination compared to controls. The dentin permeability results obtained before and after oxalate pretreatment showed that oxalate decreased dentin permeability by 98% (p 0.05) compared to acid-etched controls. This prevented outward fluid movement during bonding, resulting in better resin sealing of dentin due to the formation of a double seal of resin tags over calcium oxalate crystals in the tubules. Conclusion: Outward dentinal fluid flow may contaminate hybrid layers during adhesive bonding procedures. Pretreatment of acid-etched dentin with 3% oxalic acid prior to bonding procedures can prevent outward fluid flow during bonding and water contamination of the hydrophobic hybrid layers. Keywords: water contamination, two-photon confocal microscopy, ethanol-saturated dentin, hydrophobic hybrid layer, dentin permeability