Benzoxazinoids are a group of naturally occurring bioactive allelochemicals mostly found in cereal plants. In addition to their allelopathic effects, benzoxazinoids contain a range of health-protecting effects and pharmacological properties. The presence of these chemicals in mature cereal grains and bakery products was recently identified, making them interesting compounds from a nutritional point of view. Rye grain is the major source of dietary benzoxazinoids followed by wheat. The objective of these studies was to evaluate the uptake, distribution and metabolism of dietary benzoxazinoids in mammals. We fed a benzoxazinoid-containing rye bread-based diet to pigs (n=6), rats (n=6), and humans (n=19) and analyzed the content of benzoxazinoids and their potential metabolites in plasma, urine, bile and faeces by LC-MS/MS using electrospray ionization and multiple reaction monitoring mode. The most dominant dietary benzoxazinoid, 2-β-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc), was apparently reduced to 2-β-Dglucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), the most dominant circulating benzoxazinoid in the plasma in all three experiments. Eight different benzoxazinoids and their derivatives including some glucuronide and sulfate conjugates were excreted through the urine, HBOA-Glc being the most dominant urinary benzoxazinoid. Interestingly, 2-amino-3H-phenoxazin-3-one, a potent antibiotic, was also detected in the bile and urine. These results indicate that benzoxazinoids could be one of the key ingredients contributing to the overall health-protecting effects of whole-grain consumption.