Novel mRNA isoforms encoding the enamel matrix proteins amelin-1, amelin-2 and ameloblastin have been recently described. We have applied detailed immunohistochemical as well as non-radioactive in situ hybridization analyses to follow amelin-1 expression in developing rat incisors and molars. We constructed an expression vector, overproduced recombinant amelin in Escherichia coli and prepared an antibody. In addition to the previously reported amelin mRNA expression patterns in ameloblasts, the amelin message was also detected in pulpal mesenchymal cells including preodontoblasts and young odontoblasts. The signal in these cells persisted until deposition of mantle dentin became evident. The immunolocalization of amelin-1 in preodontoblasts and ameloblasts essentially followed the pattern of mRNA expression. The most intense staining was found in the enamel matrix adjacent to secretory ameloblasts. Focal accumulations of immunoreactive material were found at the dentinoenamel junction during the maturation stage. Also, using 5'-RACE (Rapid Amplification of cDNA Ends) we could confirm only amelin-1 and ameloblastin messages in the total RNA pool from rat molars and conclude that amelin-2 is a truncated form of ameloblastin. The sequential expression of amelin in mesenchymal and epithelial cells suggests it plays a role in cell differentiation during early tooth development.