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In order to use bovine dentin instead of human dentin for in vitro adhesion and cytotoxicity tests the permeability characteristics of human and bovine dentin should be similar. In the present study hydraulic conductance (Lp) and diffusional water flux (Js) of human and bovine dentin slices were compared. The permeability experiments were performed in a split chamber using tritiated water in physiological saline. Lp and Js of bovine dentin were 0.7- to 2.4-fold and 1.1- to 3.5-fold that of human dentin (not statistically significant). For human and bovine dentin Lp and Js increased with etching and showed an inverse linear relationship (r2 ≥ 0.7) with dentin thickness. The variability of bovine data was low (perfusion = 30%, diffusion = 22%) and about half that of the human data. In conclusion bovine dentin near the cementoenamel junction seems to be a suitable alternative for coronal human dentin for in vitro tests with respect to transdentinal permeability characteristics.