Aminoacylase from Euglena gracilis was purified by gel filtration and DEAE-Sephacel 230 times and analyzed with respect to substrate specificity, effector behaviour, pH optimum, Michaelis constant, electrophoretic mobility and molecular weight. The molecular weight is about 252000 (gel filtration). The properties of the aminoacylase from Euglena gracilis and the acylases I (EC 3.5.1.14) from the human and the pig kidneys were compared. The enzyme could be distinguished immunologically from the renal aminoacylases. No indications for the existence of a separate acylase III or of a deformylase were found.