Co- and post-translational amino-terminal processing of proteins is one mechanism by which intracellular proteins can be either protected from or targeted to degradation by the N-end Rule pathway (Bachmair, A., Finley, D., and Varshavsky, A. (1986) Science 234, 179-186). A novel enzyme, protein NH2-terminal asparagine amidohydrolase, which can function in this pathway by potentially directing critical regulatory proteins possessing an amino-terminal asparagine residue formed from the removal of N-acetylmethionine, has recently been purified and characterized (Stewart, A.E., Arfin, S. M., and Bradshaw, R. A. (1994) J. Biol. Chem. 269, 23509-23517). Here, we report the isolation and characterization of a cDNA for porcine protein NH2-terminal asparagine amidohydrolase, which indicates that it is a new type of enzyme, not homologous to any previously identified protein. This provides strong evidence for the importance of regulated protein degradation in cellular functioning.