As early as the beginning of the twentieth century some data indicated that macromolecules are able to cross the intestinal mucosa to reach the blood. Further evidence was added over the years; however, pathways for this transport still remain to be established. We report here the transfer of two pancreatic enzymes, amylase and lipase, from the intestinal lumen to the blood. Both are present in higher concentrations in the intestinal mucosa and in blood of fed rats. Upon cholinergic stimulation of pancreatic secretion, there was not only an increase in blood enzyme concentrations, but evidence for internalization by duodenal enterocytes was obtained. Following insertion of fluorochrome-tagged amylase and lipase into the duodenal lumen of fasting rats, blood and intestinal tissues were sampled at different time points. Serum activities for both enzymes clearly increased with time. Light microscopy established internalization of both proteins by duodenal enterocytes, and immunogold outlined the pathway taken by both proteins across the enterocytes. From the intestinal lumen, enzymes are channeled through the endosomal compartment to the Golgi apparatus and to the basolateral membrane reaching the interstitial space and blood circulation. Transcytosis through the intestinal mucosa thereby represents an access route for pancreatic enzymes to reach blood circulation.