It is essential to have a thorough knowledge of the bioavailability and metabolism of dietary flavonols to understand their role in disease prevention. Lightly fried onions containing 275 micromol flavonols, principally quercetin-4'-glucoside and quercetin-3,4'-diglucoside, were fed to healthy human volunteers and plasma and urine were collected over a 24 h period. Samples were analysed by HPLC with diode array and tandem mass spectrometric detection. Five flavonol metabolites, quercetin-3'-sulphate, quercetin-3-glucuronide, isorhamnetin-3-glucuronide, a quercetin diglucuronide and a quercetin glucuronide sulphate, were detected in plasma in quantifiable amounts with trace quantities of six additional quercetin metabolites. Sub-micromolar peak plasma concentrations (Cmax) of quercetin-3'-sulphate, quercetin-3-glucuronide, isorhamnetin-3-glucuronide and quercetin diglucuronide were observed 0.6-0.8 h after ingestion. In contrast, the Cmax of quercetin glucuronide sulphate was 2.5 h. The elimination half-lives (t1/2) of quercetin-3'-sulphate, quercetin-3-glucuronide and quercetin diglucuronide were 1.71, 2.33 and 1.76 h respectively, while the t1/2 of isorhamnetin-3-glucuronide was 5.34 h and that of quercetin glucuronide sulphate was 4.54 h. The profile of metabolites excreted in urine was markedly different to that of plasma with many of the major urinary components, including quercetin-3'-glucuronide, two quercetin glucoside sulphates and a methylquercetin diglucuronide, absent or present in only trace amounts in the bloodstream indicative of substantial phase II metabolism. Total urinary excretion of quercetin metabolites was 12.9 micromol, corresponding to 4.7 % of intake. If these samples had been subjected to hydrolysis, as in many previous studies, only quercetin and isorhamnetin would have been detected and quantified. The bioactivity of these metabolites should be considered.