The position of conjugation of the flavonoid quercetin dramatically affects biological activity in vitro, therefore it is important to determine the exact nature of the plasma metabolites. In the present study, we have used various methods (HPLC with diode array detection, LCMS, chemical and enzymic synthesis of authentic conjugates and specific enzymic hydrolysis) to show that quercetin glucosides are not present in plasma of human subjects 1.5 h after consumption of onions (a rich source of flavonoid glucosides). All four individuals had similar qualitative profiles of metabolites. The major circulating compounds in the plasma after 1.5 h are identified as quercetin-3-glucuronide, 3'-methyl-quercetin-3-glucuronide and quercetin-3'-sulfate. The existence of substitutions in the B and/or C ring of plasma quercetin metabolites suggests that these conjugates will each have very different biological activities.