Abstract A single triacylglycerol lipase (EC 3.1.1.3) containing approx. 420 amino acid residues has been purified from human pancreatic juice with a 30% overall yield. The method involved column chromatography on Sephadex G-25, diethylaminoethyl-cellulose, carboxymethyl-cellulose and Sephadex G-75. The molecular weight of the enzyme was estimated to be 46 000 from the amino acid composition. The purified preparation was incapable of catalyzing a prolonged hydrolysis of tributyrin at 25 °C, because of rapid inactivation. The addition of sodium taurodeoxycholate exerted a protective effect on lipase, an inhibitory action on lipolysis, and a shift of the optimal pH down to pH 7.5. Increasing the NaCl concentration enhanced the inhibitory effect of the bile salt, whereas bovine colipase and serum albumin prevented this effect. Only colipase was able to ensure a maximal reaction rate, both in the presence or absence of sodium taurodeoxycholate. Our data indicate that each molecule of human triacylglycerol lipase can easily react with one molecule of bovine colipase, and less easily with two or more molecules.