Previously, we have shown that, in rats that have had bile-pancreatic juice (BPJ) diverted from the proximal small intestine for 7 days, the exocrine pancreatic secretion was enhanced after they were fed a casein, fat-free diet. This demonstrates that the pancreatic secretion is stimulated by dietary protein with a pancreatic protease-independent pathway. To examine the chemical structure of casein responsible for the enhancement of pancreatic secretion, we prepared chemically modified casein in which lysine residues were guanidinated. Secretion of protein, amylase, and chymotrypsin in the chronic BPJ-diverted rat was increased much more after the rats were fed a diet containing guanidinated casein (250 g/kg diet) than after they were fed a diet containing intact casein (250 g/kg diet). In normal rats whose diverted BPJ was returned to the duodenum, the increases in the pancreatic secretion after consuming the guanidinated casein diet were comparable to those after consuming the intact casein diet. In vitro digestibility of guanidinated casein by trypsin and chymotrypsin was much lower than that of intact casein. Also, guanidinated casein inhibited tryptic hydrolysis of benzoyl-L-arginine p-nitroanilide to a lesser extent than did intact casein as determined by an in vitro assay. These results demonstrate that guanidinated casein is less sensitive to trypsin than is intact casein and that the structure that is sensitive to trypsin is not involved in the stimulation of pancreatic secretion in diverted rats. The results evidence that masking luminal trypsin activity does not predominantly contribute to the enhancement of pancreatic secretion in 7-day BPJ-diverted rats. Also, in normal rats, the luminal protease-independent mechanism may play a role partly in increasing the pancreatic secretion by dietary protein.