Amelogenin is the major protein of the developing enamel. Two additional exons, termed 8 and 9, have been characterized in the rat. Our aim was: to identify the mouse amelogenin exons 8/9 sequences; to investigate the potential presence of the alternative spliced isoforms of amelogenin exons 8/9; and to immunolocalize proteins containing sequences encoded by exons 8/9 during odontogenesis. RT-PCR analysis with exon 9 antisense primer generated 2 major amplicons with the use of a mouse tooth cDNA library and dental cell lines. DNA sequence analysis showed 93% identify with the rat exons 8/9 sequence. Alternative splicing of exon 3 was also found, but only in cDNAs lacking exons 8 and 9. Immunohistochemistry localized exons 8/9-encoded proteins in ameloblasts, young odontoblasts, and stratum intermedium cells. Analysis of our data supports the hypothesis that: (1) AMELX contains 2 additional exons; (2) ameloblasts and odontoblasts synthesize amelogenin 8/9; and (3) amelogenin splice variants may have unique functions during tooth formation.