The B-ring substitution pattern of flavonols is a significant structural feature for their function as free radical scavengers and antioxidants. In this paper, four differently substituted B-ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin) and a flavonol glycoside (quercitrin) were studied for their ability to bind BSA by quenching the protein intrinsic fluorescence. From the spectra obtained, the biomolecular quenching constants, the apparent static binding constants, and the binding site values were calculated. The B-ring hydroxylation of flavonols significantly affected the binding/quenching process; in general, the binding affinity increased with the number of hydroxyl groups on the B-ring. The binding constants ( Ka) were determined as myricetin (4.90 x 10(8) L/mol) > quercetin (3.65 x 10(7) L/mol) > kaempferol (2.57 x 10(6) L/mol) > galangin (6.43 x 10(5) L/mol). The glycoside substitute at the C-ring position decreased the binding affinity. The chromatographic retention factor ( K') and logarithms of apparent partition coefficient (log Kow) were linear to the logarithms of apparent binding constants (log Ka) for flavonols with increasing hydroxyl groups on the B-ring. These results showed that the hydrogen bond force play an important role in binding flavonols to BSA. These results are also in agreement with the generally accepted structure-dependent free radical scavenger and antioxidant abilities of flavonols.