The purpose of this study was to evaluate the effect of mechanical load cycling and tubule orientation on the ultimate tensile strength of crown and root dentin. Twenty bovine teeth were cleaned and their incisal surfaces reduced, resulting in a flat surface 4 mm above the cementoenamel junction. The teeth were divided into 4 groups: G1 = control (no mechanical load cycling); G2 = 1,000,000 load cycles at 50 N; G3 = 1,000,000 load cycles at 100 N; G4 = 1,000,000 load cycles at 200 N. Sections of 0.5 ± 0.1 mm thick were cut mesiodistally and the slabs further trimmed to 0.5 ± 0.1 mm in an hourglass shape, according to the tubule orientation (parallel and/or perpendicular to the long axis of the tooth) in the root and crown, with a fine diamond bur to produce a cross-sectional surface area of 0.25 mm2. All specimens were then subjected to ultimate tensile strength testing at a crosshead speed of 1 mm/min. Means and standard deviations were expressed in MPa. The bond strength data were analyzed with three-way ANOVA and Fisher's PLSD test (p 0.05). Ultimate tensile strength values did not differ statistically significantly between the loaded group and the control. However, statistical differences existed between root and coronal dentin (p 0.001) and between parallel and perpendicular orientation of tubules (p 0.001), where root and sections parallel to the tubules presented higher values than coronal and perpendicular, respectively. These differences were not related to mechanical loading. There was no effect of in vitro load cycling on ultimate tensile strength of dentin. Differences presented between ultimate tensile strength of crown vs root dentin and parallel vs perpendicular to the tubule orientation can be influenced by differences in the amount of organic and inorganic content as well as composition of the dentin matrix. It is important to consider these aspects when predicting the quality and durability of restorations according to the types of dentin and tubule orientation to be restored. Keywords: dentin, tubule orientation, mechanical load cycling, ultimate tensile strength