We previously reported that rats receiving total parenteral nutrition (TPN) undergo significant pancreatic atrophy characterized by reduced total protein and digestive enzyme expression due to a lack of intestinal stimulation by nutrients (Baumler MD, Nelson DW, Ney DM, Groblewski GE. Am J Physiol Gastrointest Liver Physiol 292: G857-G866, 2007). Essentially identical results were recently reported in mice fed protein-free diets (Crozier SJ, D'Alecy LG, Ernst SA, Ginsburg LE, Williams JA. Gastroenterology 137: 1093-1101, 2009), provoking the question of whether reductions in pancreatic protein and digestive enzyme expression could be prevented by providing amino acids orally or by intravenous (IV) infusion while maintaining intestinal stimulation with fat and carbohydrate. Controlled studies were conducted in rats with IV catheters including orally fed/saline infusion or TPN-fed control rats compared with rats fed a protein-free diet, oral amino acid, or IV amino acid feeding, all with oral carbohydrate and fat. Interestingly, neither oral nor IV amino acids were sufficient to prevent the pancreatic atrophy seen for TPN controls or protein-free diets. Oral and IV amino acids partially attenuated the 75-90% reductions in pancreatic amylase and trypsinogen expression; however, values remained 50% lower than orally fed control rats. Lipase expression was more modestly reduced by a lack of dietary protein but did respond to IV amino acids. In comparison, chymotrypsinogen expression was induced nearly twofold in TPN animals but was not altered in other experimental groups compared with oral control animals. In contrast to pancreas, protein-free diets had no detectable effects on jejunal mucosal villus height, total mass, protein, DNA, or sucrase activity. These data underscore that, in the rat, intact dietary protein is essential in maintaining pancreatic growth and digestive enzyme adaptation but has surprisingly little effect on small intestinal mucosa.