Protein methylesterase, an enzyme that hydrolyzes protein methyl esters, has been purified. The purification procedure includes ammonium sulfate and acid precipitations, and chromatographies on Sephadex G-100, Polybuffer exchanger 94, and matrex gel Green A. With this procedure, protein methylesterase was purified 1190-fold with a 28% recovery in activity. Its molecular weight has been estimated by polyacrylamide gel electrophoresis under denaturing conditions and by molecular sieving under native conditions at 31,000. Protein methylesterase has an optimum pH of 4.0 and an isoelectric point of 4.45. The enzyme is stable over a wide range of pH (2-10). The Km for ovalbumin methyl esters was estimated at 5.9 microM. Monovalent ions had little effect on activity while divalent ions at concentrations above 50 mM inhibited protein methylesterase activity in a concentration-dependent manner.