A B S T R A C T Hog pancreas was subfractionated and assessed for its ability to correct vitamin B12 malabsorption in patients with pancreatic dysfunction and in rats with partial pancreatic extirpation. The constituent obtained from the pancreas that increased vitamin B12 absorption in both humans and rats was soluble at 50,000 g, heat labile, acid stable, and approximately 20,000-25,0000 in molecular weight. The active subfractions contained tryptic and chymotryptic but no amylase or lipase activity. Thrice-crystallized trypsin corrected the vitamin B12 malabsorption in both patients with pancreatic insufficiency and in rats with subtotal pancreatectomy. These data indicate that pancreatic proteolytic enzymes-in particular, trypsin-are necessary for optimal vitamin Bn absorption.