Background: Although there are several published estimates of the endogenous amino acid composition of ileal digesta in humans, to our knowledge, there are no systematic studies of ileal digesta endogenous proteins. Objectives: We determined the nature and composition of endogenous nitrogen-containing substances lost from the upper digestive tract of humans. Design: Digesta were collected from the terminal ileum for a period of 8 h by using a nasoileal tube in 6 adult subjects fed a single meal that contained 22% of casein as the only source of nitrogen. Results: The total nitrogen that passed the terminal ileum was 39.3 mg/g native digesta dry matter. Of this amount, 86% was proteinaceous, similar to 60% was bacterial protein, similar to 7% was soluble-free protein, similar to 15% was mucin protein, and similar to 5% was protein from intact mucosal cells. For nonprotein nitrogen, similar to 5% of the total nitrogen was ammonia, and similar to 4% of the total nitrogen was urea. Bacterial and human mucosal cellular DNA nitrogen were collectively similar to 0.5% of the total nitrogen. Approximately 30% of the nonprotein nitrogen (4% of the total nitrogen) remained unidentified. This amount was assumed to include free amino acids, RNAs, amines, and the tetrapyrroles bilirubin and biliverdin. Bacterial nitrogen, combined with ammonia and urea nitrogen, represented >68% of total ileal nitrogenous losses. Conclusions: Findings are presented on the endogenous nitrogen-containing compounds that left the terminal ileum. Of particular significance is the observation that mucin was the most abundant truly endogenous component within the terminal ileal digesta. Bacterial protein, which was strictly nondietary rather than endogenous, contributed the highest proportion, by far, of nondietary protein, the result of which makes a significant contribution to published estimates of ileal endogenous amino acids and protein. The high concentration of bacterial protein and the presence of ammonia and urea nitrogen indicate potentially substantial microbial activity within the human distal small intestine. Am J Clin Nutr 2012;96:508-15.