Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37 degrees, 40 degrees, 45 degrees, and 50 degrees C for 24 hr. It was observed that the enzyme heated at 45 degrees C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45 degrees C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40 degrees C for 24 hr, the heated enzyme at 45 degrees C for 24 hr could not be reactivated when it was kept at 4 degrees C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45 degrees C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.