Four L-leucine-β-napthylamide (LβNA) hydrolyzing activities were studied in the rat CNS. Three LβNA activities were resolved using a method developed with non-denaturing polyacrylamide gel electrophoresis (PAGE) in a continuous Tris/Glycine buffer system at pH 8.5. The major activity peak of these three was studied in detail and found to have a Km of 1.34 ± 0.54 μM and a Vmax of 447.8 ± 141.2 pmol/min/mg protein for hydrolysis of LβNA and a molecular weight of either 115 or 58 kDa on 2-dimensional PAGE with SDS in the second dimension. The distribution of the three activity peaks separated by non- denaturing PAGE was studied among eight areas of the rat brain and was found to be almost homogeneous. An unusual pattern was found in trigeminal nerve where the three peaks had different activities from those found in brain. The subcellular distribution of the three peaks was also studied. All three activity peaks were found predominantly in the soluble fraction with some found in the synaptosomal fraction. The three peaks as separated by non-denaturing PAGE were later resolved by Ion Exchange Chromatography in DEAE-Sephacel under neutral conditions into four peaks, two of which corresponded to the minor peaks found on PAGE and the other two corresponded to two parts of the major peak on PAGE. The four peaks were found to differ from each other in their hydrolysis of LβNA and other aminoacyl-β-naphthylamides, and in molecular weight. One of the four peaks had similar properties to the previously characterized enzyme, soluble enkephalin degrading aminopeptidase. Improvements to the non-denaturing PAGE method are proposed to enable it to be used to resolve the four peaks and thus emphasising the validity of this sensitive method.