N-Acetylglutamate synthase, the first enzyme of arginine biosynthesis, was stabilized in crude extracts from Escherichia coli. At 4 degrees the enzyme lost less than 5% of activity per day. L-Arginine repressed the formation of N-acetylglutamate synthase. Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured. No activity (i.e. less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cell cultivated on minimal medium. In a 6-fold purified preparation L-arginine inhibited the enzyme. Of 11 precursors and analogues of arginine tested only O-[L-norvalyl-5]-isourea inhibited N-acetylglutamate synthase as strongly as L-agrinine.