Amelogenins comprise as much as 90% of the protein in the developing enamel matrix. Separating amelogenins by gel electrophoresis reveals a complex of polypeptides with apparent mobilities ranging from low molecular weight species on up to 28,000 Daltons. A major objective of our research is determine the extent to which alternative RNA splicing contributes to this heterogeneity. We have cloned seven alternatively spliced mouse amelogenin mRNAs. The predicted translation products of these messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. The 194 residue amelogenin is the only mouse amelogenin to include a polypeptide segment encoded by exon 4, which has a deduced amino acid sequence of KSHSQAINTDRTAL. Antibodies were raised against synthetic exon 4 encoded polypeptides and used to immunostain histologic tooth sections. These data indicate that alternatively spliced amelogenin mRNAs are translated into protein and secreted into the enamel matrix.