The mechanism of the relaxant action and the structure-activity relation of flavonols (fisetin, quercetin, and 3,3',4'-trihydroxyflavone) and flavones (apigenin, chrysin, and luteolin) were examined in rat isolated thoracic aorta. The control responses to flavonols and flavones were compared with responses observed after the removal of the endothelium or in the presence of the L-type Ca2+ channel blocker, nifedipine (10(-7) M). The effects of flavonoids on contraction caused by the influx of extracellular Ca2+ and agonist-induced release of intracellular Ca2+ also were investigated. The flavones exhibited endothelium-independent vasorelaxation, whereas the removal of the endothelium significantly decreased the sensitivity of the relaxant responses to the flavonols without affecting the maximal relaxation. In the presence of nifedipine, the responses to apigenin, luteolin, and quercetin were significantly inhibited, but relaxation to chrysin, fisetin, and 3,3',4'-trihydroxyflavone was unaffected. All flavonols and flavones caused concentration-dependent inhibition of the contractile responses to exogenous application of Ca2+ and the release of intracellular Ca2+ stimulated by phenylephrine. Of the six flavonoids examined, 3,3',4'-trihydroxyflavone was the most potent when causing vasorelaxation or inhibition of contraction caused by the influx or release of Ca2+. In conclusion, these studies provide evidence that the hydroxyl substitution in the carbon 3 position that characterizes the flavonols is important in stimulating endothelium-dependent vasorelaxation, and the absence of hydroxyl substitution on the A phenolic ring enhances the relaxant action.