The regional, cellular and subcellular distribution of neutral proteinase hydrolyzing serum albumin in the brain was studied. The enzyme was detected in soluble cell fraction. The neutral proteinase activity in the neuron cell enriched fraction from the cat cortex was 4 times as high as that in the glia. Neutral proteinase was isolated and purified from bovine cerebellum by ammonium sulfate precipitation, dialysis, gel chromatography and affinity chromatography on albumin-Biogel P-300. Two peaks of neutral proteinase activity were observed. The degree of purification was 3400 and 7700, respectively; the recovery was 6 and 4%, respectively. The enzyme was activated by 2-mercaptoethanol and was inhibited by EDTA and p-chloromercurybenzoate. Phenylmethylsulfonylfluoride and pepstatin had no effect on the enzyme activity. The inhibitory analysis demonstrated that the enzyme probably contains some proteinases capable to bind to immobilized serum albumin.