Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a β-glucuronidase inhibitor). Furthermore, pure recombinant human β-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (kcat/Km=1.3×103 M-1 s-1 for equol-7-O-glucuronide and 26×103 M-1 s-1 for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal β-glucuronidase from various human cells.