Glucosamine-6-phosphate synthetase (G6P synthetase) inactivator was purified from rat submandibular gland by a combination of Sephadex G-100 gel filtration and column isoelectric focusing. The purified inactivator showed a single protein band by polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 26, 000 by gel chromatography. It caused an apparent inactivation of G6P synthetase partially purified from rat liver. The purified inactivator was found to be a trypsin-like enzyme because it specifically hydrolyzed the trypsin substrate, Nα-benzoyl-L-arginine-p-nitroanilide, and was inhibited by soybean trypsin inhibitor. Thus, the action of G6P synthetase inactivator may be due to the degradation of the synthetase through the proteolytic activity of the inactivator.