1. 1.|Preparations of purified pig kidney aminoacylase ( N- Acylamino-acid amidohydrolase, EC 3.5.1.14) were obtained by Sephadex and DEAE-cellulose chromatography in homogeneous form as judged by polyacrylamide gel electrophoresis and immunoelectrophoresis. 2. 2.|The apparent molecular weight of the enzyme, determined by gel filtration, was about 86 000. After treatment with mercaptoethanol, performic acid or sodium dodecyl sulphate a band with an apparent molecular weight of approximately 43 000 was observed in polyacrylamide gels containing sodium dodecyl sulphate. Thus pig kidney aminoacylase seems to be composed of two subunits. 3. 3.|The amino acid composition of the enzyme was determined. Aminoacylase contains 772 amino acids, which corresponds to a molecular weight of 85 500. 12 tryptophan and 12 half-cystine residues were found. 4. 4.|Each subunit of the enzyme contains two -SH groups of different reactivity and two disulfide bonds one of which is easily cleaved by -SH compounds, the second only by performic acid oxidation. 5. 5.|Chemical modification of two -SH groups abolishes the catalytic activity of aminoacylase. Cleavage of two disulfide bonds also inactivates the enzyme. it is suggested that the enzyme has two active sites each containing an essential -SH group and disulfide bond. One active site is assumed to be part of each subunit.