N-Acetylglutamate synthetase (EC 2.3.1.1), the first enzyme of arginine synthesis was shown to be under multiple control by the reaction products and the endproducts of the pathway in tenfold purified extracts from . Synergistic inhibition of the enzyme was exerted by N-acetyl-L-glutamate and polyamines. At 0.5 mM N-acetyl-L-glutamate spermine was the most potent inhibitor, whereas spermidine, cadaverine and putrescine inhibited the enzyme to a lesser extent. Furthermore, feedback-inhibition by L-arginine was enhanced synergistically by N-acetyl-L-glutamate and CoA.