1. Arylamidase activity was isolated from Enterolobium contortisiliquum seeds (2 U/g) using L-Leu-2-naphthylamide as substrate to monitor the purification. 2. The enzyme preparation was purified 733-fold by ammonium sulfate precipitation, and by ion exchange and gel filtration chromatography, in 6.6% yield. 3. SDS-Polyacrylamide gel electrophoresis after fast protein liquid chromatography on a Mono Q column, showed only one protein band with a molecular weight of 35 kDa. 4. The optimum pH for arylamidase activity was 6.5. Taking the hydrolysis rate of Lys-2-naphthylamide as one, the relative rates at which the other substrates were hydrolyzed were: Leu-2-naphthylamide, 30, Met-2-naphthylamide, 18, Arg-2-naphthylamide, 2, Ala-2-naphthylamide, 1.5, and L-Leu-p-nitroanilide, 26. 5. The arylamidase activity was inhibited 50% by 0.1 mM HgCl2, 0.1 mM MnCl2, 0.1 mM ZnCl2, 0.13 mM NiCl2, 0.2 mM o-phenanthroline and 1 microM sodium p-hydroxymercuribenzoate, and activated 35% by 5.0 microM EDTA. Iodoacetate (0.67 mM), dithioerythritol and 2-mercaptoethanol (3.3 mM), and chloride ions (0.2 M) had no effect on the enzyme activity.