Microstructure in terms of hierarchical assembly exists widely in mineralized biomaterials, fulfilling an important role in setting up their outstanding properties. The purpose of this study was to investigate the hierarchical assembly of enamel structure and functions, which are related to the unique characteristics of enamel. Human enamel taken from mature third molars was explored using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM) and atomic force microscopy (AFM). Integrating the microscopic observations revealed the high complexity of the well-organized enamel structure in terms of hierarchical assembly. Based on these observations, seven hierarchical levels of the microstructure were proposed and described, using a scheme representing a complete spectrum of the organization in detail, covering a range from microscale to nanoscale: hydroxyapatite crystals (Level 1) at first form mineral nanofibrils (Level 2); the nanofibrils always align lengthways, aggregating into fibrils (Level 3) and further thicker fibres (Level 4); prism/interprism continua (Level 5) are then composed of them. At the microscale, prisms assemble into prism bands (Level 6), which present different arrangements across the thickness of the enamel layer (Level 7). Analysis of the enamel and bone hierarchical structure suggests similarities of scale distribution at each level. This study also aimed to understand further the structural-mechanical relations at each hierarchical level. Copyright © 2007 John Wiley & Sons, Ltd.