Atomic force microscopy (AFM) combined with a nano-indentation technique was used to reveal the structure and to perform site-specific mechanical testing of the enamel of third molars. Nano-indentations (size<500 nm) were made in the cusp area to measure the mechanical properties of single enamel rods at different orientations. The influence of etching on the physical properties was studied and etching conditions that did not significantly alter the plastic-elastic response of enamel were defined. Elasticity and hardness were found to be a function of the microstructural texture. Mean Young's moduli of 87.5 (+/-2.2) and 72.2 (+/-4.5) GPa and mean hardness of 3.9+/-0.3 and 3.3+/-0.3 GPa were measured in directions parallel and perpendicular to the enamel rods, respectively. Analysis of variance showed that the differences were significant. The observed anisotropy of enamel is related to the alignment of fibre-like apatite crystals and the composite nature of enamel rods. Mechanical properties were also studied at different locations on single enamel rods. Compared to those in the head area of the rods, Young's moduli and hardness were lower in the tail area and in the inter-rod enamel, which can be attributed to changes in crystal orientation and the higher content of soft organic tissue in these areas.