4-Deoxynivalenol is one of the most prevalent mycotoxins in grain-based food and feed products worldwide. Conjugation of deoxynivalenol to glucuronic acid and elimination via the urine appears to be the major metabolism pathway, although with differing efficiency in different species. In order to make pure deoxynivalenol glucuronides for analytical methodologies available we intended to enzymatically synthesize glucuronides of deoxynivalenol using rat and human liver microsomes supplemented with uridine 5'-diphosphoglucuronic acid and alamethicin as detergent. Three glucuronides were isolated and purified using solid-phase extraction of microsomal incubations and subsequent semipreparative hydrophilic interaction chromatography. NMR spectra were obtained for all three compounds from solutions in methanol, showing that deoxynivalenol 3-O-β-D-glucuronide and deoxynivalenol 15-O-β-D-glucuronide were the major products from incubations of deoxynivalenol with rat and human liver microsomes, respectively. The NMR spectra of a third glucuronide showed replacement of the C-8 carbonyl by a ketal carbon. This glucuronide was finally identified as deoxynivalenol 8-O-β-D-glucuronide. The present study provides unequivocal structural evidence for three glucuronides of deoxynivalenol formed by liver enzymes.