Abstract A new protease which specifically acts on the apo-form of pyridoxal enzymes was discovered in rat small intestine and skeletal muscle. This enzyme was purified about 500-fold from the small intestine of vitamin B6-deficient rats. Some proporties of the enzyme and its action were studied. The enzyme split the apo-protein of ornithine transaminase into two products, a homogeneous smaller protein and an oligopeptide, and thereby inactivated the transaminase. The substrate enzyme, ornithine transaminase, and the larger product have s020,w values of 9.9 and 5.1, respectively. The enzyme inactivated all of the apo-pyridoxal enzymes tested, including serine dehydratase, ornithine transaminase, tyrosine transaminase and aspartate transaminase, but did not affect non-pyridoxal enzymes (glutamic dehydrogenase, lactic dehydrogenase, urease, and glutaminase), bovine serum albumin or some synthetic substrates. Addition of pyridoxal phosphate or a high concentration of pyridoxal had a protective effect against the specific protease, but another active coenzyme, pyridoxamine phosphate, did not. The activity of this protease in the small intestine increased during vitamin B6 deficiency. A reciprocal relation was found between the activity of this protease and that of ornithine transaminase in the small intestine.