Abstract Arylamidase (E.C.188.8.131.52) is able to split a number of amino acid arylamides, preferentially l-alanine-β-naphthylamide. Nevertheless the rate of hydrolysis of l-leucine-4-nitroanilide is not very much slower when measured, er optimal conditions. From a clinical view, there is good correlation between the two substrates in different diseases. It is concluded that there is no need for replacing the well established, kinetic arylamidase assay with l-leucine-4-nitroanilide by another test system using l-alanine-β-naphthylamide as substrate. In another series of experiments serum arylamidase was separated into subfractions by agar gel electrophoresis. The existence of a second, slower moving band of activity is claimed to be of specific value for the diagnosis of acute pancreatitis. We could not confirm these findings: in 8 cases out of 20 patients with well defined acute pancreatitis, isoenzyme 2 was absent. In contrast, it was detectable in 50% of all liver disease and in 40% of patients with slightly elevated serum activities of arylamidase or alkaline phosphatase without any symptoms of pancreatic or liver disease. It seemed moreover, that isoenzyme 2 regularly occurs in cases of cholestasis.