To create a bioceramic with unique materials properties, biomineralization exploits cells to create a tissue-specific protein matrix to control the crystal habit, timing, and position of the mineral phase. The biomineralized covering of vertebrate teeth is enamel, a distinctive tissue of ectodermal origin that is collagen-free. In forming enamel, amelogenin is the abundant protein that undergoes self-assembly to contribute to a matrix that guides its own replacement by mineral. Conserved domains in amelogenin suggest their importance to biomineralization. We used gene targeting in mice to replace native amelogenin with one of two engineered amelogenins. Replacement changed enamel organization by altering protein-to-crystallite interactions and crystallite stacking while diminishing the ability of the ameloblast to interact with the matrix. These data demonstrate that ameloblasts must continuously interact with the developing matrix to provide amelogenin-specific protein to protein, protein to mineral, and protein to membrane interactions critical to biomineralization and enamel architecture while suggesting that mutations within conserved amelogenin domains could account for enamel variations preserved in the fossil record.In mammals mesenchyme-derived bone is the dominant biomineralized tissue and is composed of substituted hydroxyapatite crystals with specific proteins distributed in an abundant collagen network (1-3). For bone, the mineral composite is dynamic with both the protein and mineral phases being constantly remodeled by cells. A notable exception to such a biomineralization theme is found in enamel, the vertebrate covering of teeth. In teeth, ectoderm-derived ameloblasts synthesize a short-lived, diverse repertoire of proteins that self-assemble to produce a matrix that directs its own replacement by the mineral phase (4, 5). Mature enamel is a bioceramic composed of less than 0.5% protein that is dispersed among the longest hydroxyapatite crystals in the vertebrate body (6 -8). Unlike mesenchyme-derived bone, enamel does not contain collagen, does not have the capacity of self-repair, and does not undergo remodeling. Nonetheless, enamel lasts the lifetime of the organism due its unique material properties and remarkable resistance to failure (9, 10). These properties are due in part to the cell-directed organization of crystallites into bundles called rods. Rods are further woven together during their formation by cell migration to form a unique three-dimensional lattice architecture that resists wear and deformation (9 -14).Amelogenin is the most abundant of the enamel matrix proteins and undergoes self-assembly to form nanospheres (15-17). Amelogenin nanospheres appear to control the habit of hydroxyapatite crystallites by directing mineral growth to the ends (cЈ axis) of the crystallites by providing the space for crystallites to grow within and by buffering the local environment against protons liberated during mineral deposition (5, 7, 18 -20). The amino acid sequence of amelogenin is highly ...