Previously, a cDNA encoding a novel matrix metalloproteinase (enamelysin) was isolated from a porcine enamel organ-specific cDNA library. The cloned mRNA is tooth-specific and contains an open reading frame encoding a protein composed of 483 amino acids (Gene, 183:(1-2), p123-128, 1996). Here, we show that: 1) The expression of enamelysin mRNA is not limited to the enamel organ as previously reported. The enamelysin message is also expressed at very low levels in the pulp organ. 2) Northern analysis reveals that the enamelysin mRNA displays a developmentally defined pattern of expression in the enamel organ. The message is expressed at relatively high levels during the presecretory and early transition stages of development. However, during late maturation, the quantity of enamelysin mRNA is greatly reduced. Conversely, the low message levels in the pulp organ remain relatively constant throughout these developmental stages. 3) The enamelysin cDNA was ligated into a prokaryotic expression vector and recombinant enamelysin containing a His tag was purified from E. coli. Zymographic analysis utilizing recombinant murine amelogenin as the substrate, reveals that the purified enamelysin degrades amelogenin. Since enamelysin is developmentally regulated and is capable of degrading amelogenin, it is likely to play a significant role during enamel biomineralization.