We aimed to analyze the differential gene expression in various murine dental tissues, expecting to find novel factors that are involved in tooth formation. We here describe the identification of a novel ameloblast-specific gene, amelotin (AMTN), by differential display polymerase chain-reaction (DD-PCR) analysis of microdissected ameloblasts, odontoblasts, dental pulp, and alveolar bone cells of 10-day-old mouse incisors. The conceptually translated protein sequence was unique and showed significant homology only with its human orthologue. The amelotin genes from mouse and human displayed a similar exon-intron structure and were expressed from loci on chromosomes 5 and 4, respectively, which have been associated with various forms of amelogenesis imperfecta. Expression of amelotin mRNA was restricted to maturation-stage ameloblasts in developing murine molars and incisors. Amelotin protein was efficiently secreted from transfected cells in culture. Taken together, our findings suggest that amelotin is a novel factor produced by ameloblasts that plays a critical role in the formation of dental enamel.