The reaction of nitrite with different amino acids containing secondary amino groups was tested under simulated in-vitro conditions of the digestive tract. After treatment, tryptophan was the only amino acid that exhibited specific UV absorbance of nitrosamines at 335 nm, supporting the assumption that it is the main source of endogenous nitrosamines. The combined effect of pH (from 2 to 6.5) and nitrite (from 0.1 to 20 mM) was analyzed and the mechanisms and kinetic laws of tryptophan N-nitrosation were determined. The model was then completed by the addition of iron and various antioxidants in concentrations reflecting different diets. The results clearly demonstrated that, in the presence of iron, large amounts of N-nitroso-tryptophan can be formed even at neutral pH, as in the intestine. Antioxidants (ascorbic acid, trolox C, β carotene, chlorogenic acid, phytic acid and butylated-hydroxytoluene) had various impacts on the extent of N-nitrosation, depending on the iron level.