In recent years, the frequent detection of the banned substance thiouracil (TU) in livestock urine has been related to its endogenous formation following consumption of glucosinolate-rich Brassicaceae crops. Besides, TU residues have been recovered in these crops upon plant-derived myrosinase hydrolysis. Through in vitro bovine and porcine static digestive simulations, the influence of gastrointestinal digestion of Brassicaceae-derived matrixes on TU formation was assessed. Following derivatization and LC-MS(2) analysis, TU was detected in colonic suspensions with traditional rapeseed, coarse colza "00" meal, cauliflower, and broccoli ranging from 3.47 to 30.96 μg kg(-1) (bovine) and from 3.55 to 26.34 μg kg(-1) (porcine). In stomach and small intestinal fluids, TU remained unfound, whereas upon rumen simulation TU was detected for coarse colza "00" meal (4.43 μg kg(-1)) and grounded traditional rapeseed (0.35 μg kg(-1)). The origin of this detection was investigated through filter-sterilizing and autoclaving the fecal inoculum causing a significant decrease in TU concentration, thereby reinforcing the possibility of an active bacterial involvement, which however was characterized with a high interanimal variation. In conclusion, these results support the previously proven endogenous origin of TU and acknowledge the active role of the gastrointestinal bacteria in TU formation, through production of an extracellular component.