We studied the distribution of aminoacylase, an enzyme catalyzing the hydrolysis of N-acylamino acids, in thermophilic bacteria, and found Bacillus thermoglucosidius DSM 2542 to be the best producer of the enzyme. The enzyme, purified 13, 400-fold to homogeneity in an overall yield of 34%, has a molecular weight of about 175, 000, and is composed of four subunits identical in molecular weight (43, 000). The enzyme contains 4 g atoms of zinc per mol of enzyme protei-n. The enzyme catalyzes hydrolysis of various kinds of N-acyl-L-amino acids with very high molecular activity compared to those of fungal and mammalian enzymes: Vmax and Km for N-acetyl-Lmethionine are 3410 units/mg protein and 7.9 mM, respectively. Great stability at high temperatures and with organic solvents and protein denaturants is a characteristic of the enzyme.