Although pancreatic enzymes clearly degrade R binder, a nonintrinsic factor binder, the full scope of the pancreatic role in cobalamin absorption remains the subject of debate. Therefore the direct effect of pure human pancreatic juice (PPJ) on ileal cobalamin absorption in the absence of intrinsic factor was studied. PPJ significantly enhanced cobalamin uptake in guinea pig ileal loop perfused in vivo. It did not do so in the jejunum. This PPJ activity in the ileum was further stimulated by enteropeptidase and inhibited by aprotinin. The intestinal mucosa remained intact during our study by morphologic and inulin clearance criteria and behaved normally with respect to intrinsic factor and nonintrinsic factor binders. Since no intrinsic factor was present in the perfusate, PPJ must directly enhance cobalamin uptake by the ileum, perhaps promoting cobalamin attachment to receptor sites for subsequent transport by intrinsic factor. PPJ thus seems to affect cobalamin absorption at several levels. Previous studies have established its interaction with luminal R binders and with bile. The findings now indicate that pancreatic juice may have an additional, more direct role in promoting cobalamin absorption in the ileum.