The three-dimensional morphology of human tooth fissures and the quantification of mineral distribution in fissure enamel are pertinent to the development and diagnosis of caries. Synchrotron X-ray microtomography was used to measure linear attenuation coefficients (at 25 keV) at high spatial resolution with a volume-imaging element (cubic voxel) of 4.9 × 4.9 × 4.9 μm3 in a block from a human premolar that included part of a stained fissure. From the linear attenuation coefficient, the mineral concentration, expressed as gHAp cm−3 (where HAp is stoichiometric hydroxyapatite), was calculated. The mean mineral concentration in bulk enamel was 2.84 gHAp cm−3. Well-defined regions (1.5–2.6 gHAp cm−3), extending up to ≈ 130 μm from the base of some narrower lengths of the fissure and up to ≈ 50 μm deep from the fissure surface, were attributed to hypomineralization. Other regions of low mineral concentration, some (1.4–2.3 gHAp cm−3) lying within the expected course of the fissure base and some (2.2–2.7 gHAp cm−3) deep to the pit, were also considered to be of developmental origin. However, a diffuse distribution of low mineral concentrations (2.2–2.7 gHAp cm−3) in the pit walls was attributed primarily to demineralization from caries. The fissure contained heterogeneous material (≤ 0.5 gHAp cm−3) exhibiting partial mineralization.