Abstract Protein l -isoaspartate ( d -aspartate) O-methyltransferase (EC 126.96.36.199) is a repair enzyme that methylates abnormal l -isoaspartate residues in proteins which arise spontaneously as a result of aging. This enzyme initiates their conversion back into the normal l -aspartate form by a methyl esterification reaction. Previously, partial cDNAs of this enzyme were isolated from the higher plant Arabidopsis thaliana. In this study, we report the cloning and expression of a full-length cDNA of l -isoaspartyl methyltransferase from A. thaliana into Escherichia coli under the P BAD promoter, which offers a high level of expression under a tight regulatory control. The enzyme is found largely in the soluble fraction. We purified this recombinant enzyme to homogeneity using a series of steps involving DEAE–cellulose, gel filtration, and hydrophobic interaction chromatographies. The homogeneous enzyme was found to have maximum activity at 45°C and a pH optimum from 7 to 8. The enzyme was found to have a wide range of affinities for l -isoaspartate-containing peptides and displayed relatively poor reactivity toward protein substrates. The best methyl-accepting substrates were KASA- l -isoAsp -LAKY ( K m = 80 μM) and VYP- l -isoAsp -HA ( K m = 310 μM). We also expressed the full-length form and a truncated version of this enzyme (lacking the N-terminal 26 amino acid residues) in E. coli under the T7 promoter. Both the full-length and the truncated forms were active, though overexpression of the truncated enzyme led to a complete loss of activity.