Genome sequencing and gene mapping have permitted the identification of HEVIN (SPARC-Like1) as the probable ancestor of the enamel matrix proteins (EMPs), amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). We have undertaken a phylogenetic analysis to elucidate their relationships. AMEL genes available in databases, and new sequences obtained in BLAST searching genomes or expressed sequence tags, were compiled (22 full-length sequences), aligned, and the ancestral sequence calculated and used to search for similarities using PSI-BLAST. Hits were obtained with the N-terminal region of AMBN, ENAM, and HEVIN. We retrieved all available AMBN (n = 8), ENAM (n = 3), and HEVIN (n = 4) sequences. The sequences of the four proteins were aligned and analyzed phylogenetically. AMEL and AMBN are sister genes, which diverged after duplication of a common ancestor issued from ENAM. The latter derived from a copy of HEVIN. Comparisons of gene organization, amino acid sequences and location of ENAM and AMBN, adjacent on the same chromosome, suggest that AMBN is closer to ENAM than AMEL. This supports AMEL as being derived from AMBN duplication. This duplication occurred long before tetrapod differentiation, probably in an ancestral osteichthyan. The story of AMEL origin is completed as follows: SPARC→HEVIN→ENAM→AMBN→AMEL.