Multiphoton microscopy has been used to reveal structural details of dentine and enamel at the dentin-enamel junction (DEJ) based on their 2-photon excited fluores- cence (2PEF) emission and second harmonic generation (SHG). In dentine tubule 2PEF intensity varies due to protein content variation. Intertubular dentin produces both SHG and 2PEF signals. Tubules are surrounded by a thin circular zone with a lower SHG signal than the bulk dentine and the presence of collagen fibers perpen- dicular to the tubule longitudinal axis is indicated by strong SHG responses. The DEJ appears as a low inten- sity line on the 2PEF images and this was never pre- viously reported. The SHG signal is completely absent for enamel and aprismatic enamel shows a homogeneous low 2PEF signal contrary to prismatic enamel. The SHG intensity of mantle dentine is increasing from the dentine-enamel junction in the first 12 mm indicating a progressive presence of fibrillar collagen and corresponding to the more external part of mantle dentine where matrix metallo-proteases accumulate. The high information content of multiphoton images confirms the huge potential of this method to investigate tooth structures in physiological and pathological conditions. Dentine-Enamel Junction. Superposition of SHG and 2PEF images showing the relationship of enamel and dentine.