Dietary flavonoids are thought to have health benefits possibly due to antioxidant and anti-inflammatory properties. Many previous in vitro studies examining the bioactivity of flavonoids have failed to consider the effects of metabolic transformation on flavonoid activity. In this study we examined the effect of quercetin and its major metabolites on the production of pro-inflammatory eicosanoids by human leukocytes. Studies comparing free radical scavenging, antioxidant activity and eicosanoid production demonstrate that there are different structural requirements for antioxidant and anti-inflammatory activity. We also investigated the effect of metabolic transformation on flavonoid bioactivity by comparing the activity of quercetin and its major metabolites to inhibit inflammatory eicosanoid production from human leukocytes. Quercetin was a potent inhibitor of leukotriene B-4 formation in leukocytes (IC50 similar to 2 mu M), and its activity was dependent on specific structural features, particularly the 2,3-double bond of the C-ring. Functionalisation of the T-OH group with either methyl or sulfate reduced inhibitory activity up to 50% while a glucuronide substituent at the 3'-OH effectively removed the LTB4 inhibitory activity. The major quercetin metabolite quercetin-3'-O-sulfate retained considerable lipoxygenase inhibitory activity (IC50 similar to 7 mu M) while quercetin-3-O-glucuronide maintained antioxidant activity but had no lipoxygenase inhibitory activity at physiological concentrations. In conclusion, we have found that structural modification of quercetin due to metabolic transformation had a profound effect on bioactivity, and that the structural features required for antioxidant activity of quercetin and related flavonoids were unrelated to those required for inhibition of inflammatory eicosanoids. Crown Copyright (c) 2007 Published by Elsevier Inc. All rights reserved.