The existance of a new group specific protease for pyridoxal enzymes was proved in skeletal muscle of rats. The enzyme was purified until homogeneous protein judging from acrylamide gel disc electrophoresis and certain properties were studied. The molecular weight of the enzyme was calculated as about 13,000 using Sephadex G-75 column chromatography with several kinds of markers. The purified enzyme shows strict substrate specificity for pyridoxal enzyme group and the enzyme inactivates only apo-form of pyridoxal enzyme group tested and holo-form of pyridoxal enzymes are not affected. No the other types of enzyme were inactivated by the purified protease. Usual synthetic substrates for trypsin like p-toluene-sulfonyl-L-arginine methyl ester are not cleaved by the protease. N-acetyl-L-tyrosine-ethyl ester which is one of substrates for chymotrypsin was cleaved but p-aminophenyl-β-phenyl-propionate which is chymotrypsin inhibitor does not show any inhibitory action. The optimum pH of the protease exists in alkaline side at 9.0,and 10^<-4>M of Ca^<2+> ion inhibits this enzyme activity till 50%.