Under physiological conditions, L-aspartyl (L-Asp) and L-asparaginyl residues in proteins are spontaneously isomerized or racemized to D-aspartyl (D-Asp) or D,L-isoaspartyl (D,L-isoAsp) residue. These atypical Asp residues can interfere with protein activity and lead to disruption of cellular function. Protein L-isoaspartyl/D-aspartyl O-methyltransferase (PIMT) is a repair enzyme that initiates the conversion of L-isoAsp (or D-Asp) residues to L-Asp residues. PIMT-Deficient mice exhibit accumulation of L-isoAsp in several tissues and die from progressive epileptic seizures at a mean age of 42 days. However, the biological roles of PIMT are still largely unknown. To further our understanding of the function of this protein, we developed an assay to measure PIMT activity in cell lysates. Additionally, we generated PIMT-knockdown cells by stable transfection of HEK293 cells with PIMT small interfering (si) RNA. Northern blotting and immunoblot analysis revealed that PIMT mRNA and protein levels were significantly decreased in the knockdown cells. In addition, significant levels of proteins that contained isoAsp residues accumulated in these cells, and immunoblot analysis revealed that Raf-1, MEK, and ERK were hyperphosphorylated upon EGF stimulation compared to control cells. These results indicate that the ability to repair atypical Asp residues is important for normal MAP kinase signaling.