Background Endogenous amino acids (AAs) contribute to terminal ileal digesta and must be corrected for in determining coefficients of true ileal digestibility. Such estimates are also needed for the factorial calculation of AA requirements. Objective The effect of the form of delivery of dietary AAs on endogenous nitrogen and AA flows at the end of the ileum was studied. Methods Isotopically labeled 15N-casein (33–34 g), in either a hydrolyzed (HC) or intact (C) form, was included as the sole source of nitrogen in a mixed meal (320 mmol N) consumed by healthy adult humans equipped with a triple-lumen sampling tube in the small intestine. Ileal endogenous AA flows were determined by isotope dilution. An additional meal (A) containing a free AA mixture (306 mmol N) simulating the AA composition of casein was included. Serine was omitted from the AA mixture to allow direct determination of its ileal endogenous flow. Reults Endogenous N and AA flows did not differ (P > 0.05) for diets C and HC, with mean respective N flows of 728 and 617 mg/8 h (± pooled SD: 144 mg/8 h). Endogenous serine flow was similar (P > 0.05) for diets C, HC, and A [181, 169, and 191 mg/8 h (± 56 mg/8 h)]. Recycling of the 15N marker was determined to be ∼11%, suggesting that the 15N endogenous loss values could underestimate endogenous protein and AA losses by ∼6% (the proportion of recycled 15N divided by the sum of endogenous N and recycled 15N). Conclusions The 3 different forms of dietary AA delivery (free AAs, HC, or C) elicited similar ileal endogenous AA flows in the adult human. This study was registered at clinicaltrials.gov as NCT 00873951.