Flavonoids are known to modulate catalytic activity and expression of various enzymes. Glutathione S-transferases (GSTs) are the important biotransformation enzymes defending cells against potentially toxic xenobiotics. Therefore, the modulation of GST activity may influence detoxification of xenobiotics. The aim of this study was to evaluate the in vitro inhibitory activity of several dietary flavonoids towards purified equine liver cytosolic GST. Pure GST was incubated in the presence or absence of flavonoids (10 nM-100 µM), its activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate, and half maximal inhibitory concentrations (IC(50)) were determined. The obtained results were confirmed by GST activity staining of native polyacrylamide gel electrophoresis (PAGE) gels. For the most potent inhibitor, the inhibition kinetics study was performed. From 24 flavonoids tested, the most potent GST inhibitor was gallocatechin gallate (IC(50) = 1.26 µM). The inhibition kinetics of this compound followed noncompetitive mechanism versus both glutathione (K(i) = 35.9 µM) and CDNB (K(i) = 34.1 µM). The inhibitory potency of different flavonoids for GST activity depended mainly on the pattern of hydroxylation and number of hydroxyl groups in the ring B. Especially, pyrogallol-type catechins with 3-OH group esterified with gallic acid showed strong potential to inhibit GST in vitro.