The membrane-bound enzyme which catalyzes the degradation of thyrotropin-releasing hormone (TRH; Glp-His-Pro-NH2) could be released from membranes of rat and pig brain by treatment with trypsin under very mild incubation conditions. The solubilized enzyme was purified 200,000-fold, with an overall yield of 20%, by conventional chromatographic methods. The enzyme preparation appeared to be electrophoretically homogenous since SDS/PAGE analysis revealed a single band with a molecular mass of 116,000 Da. By gel-filtration chromatography, a molecular mass of 230,000 Da was estimated, suggesting that the enzyme consists of two identical subunits. The enzyme could be identified as a glycoprotein by lectin-binding analysis and by the reduction of the molecular mass to 97,000 Da upon treatment of the denatured enzyme with endoglycosidase-F/N-glycosidase F. In its native form, however, the enzyme was only partially deglycosylated and retained full enzymatic activity. In addition to TRH, the enzyme also hydrolyzed L-5-oxoprolyl-beta-naphthylamide, and thus a convenient fluorimetric assay could be established to determine high enzyme activities. The hydrolysis of both substrates was found to obey Michaelis-Menten kinetics, but considerable differences in the respective Km and Vmax values were noticed.