A glycerol-ester hydrolase has been characterized and partially purified (23-fold) from human small intestine. A pancreatic origin was excluded through the use of an immunoserum directed against human pancreatic juice, while a pharyngeal (gastric) origin was unlikely owing to the optimum pH range of activity of the gastric enzyme as compared to that of the intestinal enzyme. The glycerol-ester hydrolase is mainly active toward short-and medium-chain di- and triglycerides and toward micellar long-chain monoglycerides. A role in the absorption of neutral lipids, mainly in pathological situations, is postulated.