The aim of the present study was to compare the protein-free diet, guanidinated casein (GuC) and enzyme hydrolysed casein (EHC) methods for the quantification of endogenous amino acid (AA) flow in the avian ileum. Growing broiler chickens (5 weeks old) were used. All three assay diets were based on dextrose, and in the GuC and EHC diets GuC or EHC were the sole source of N. Endogenous AA flows determined with the use of protein-free diet were considerably lower (P>0·05) than those determined by the GuC and EHC methods. The total endogenous AA flows determined by the GuC and EHC methods were almost 3-fold greater (P>0·05) than those determined by the protein-free diet. The endogenous AA values obtained from GuC and EHC methods were similar (P<0·05), except for the flow of arginine, which was lower (P>0·05) in the EHC method. Glutamic acid, aspartic acid, threonine and glycine were the predominant endogenous AA present in digesta from the distal ileum. The contents of methionine, histidine and cystine were lower compared with other AA. The method of determination had no effect on the AA composition of endogenous protein, except for threonine, glutamic acid, lysine, arginine and cystine. The concentrations of threonine and arginine were lower (P>0·05) and that of lysine was higher (P>0·05) with the EHC method compared with the other two methods. The concentration of glutamic acid was greater (P0·05) and that of cystine was lower (P>0·05) in the EHC and GuC methods compared with the protein-free diet method. The results showed that the ileal endogenous flows of N and AA are markedly enhanced by the presence of protein and peptides, above those determined following feeding of a protein-free diet. It is concluded that the use of EHC and GuC methods enables the measurement of ileal endogenous losses in chickens under normal physiological conditions.