A series of structurally related flavonoids and related compounds were evaluated whether they have inhibitory properties on the 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 18.104.22.168, 5'-NT) activity. Some of the flavonoids tested inhibit the enzyme such as quercetin, morin, apigenin, chrysin, myricetin, luteolin, diosmetin, (+/-)naringenin and diosmin. Rutin, naringin, hyperosid, (+/-)catechin, caffeic acid and rosmarinic acid had no inhibitory effect on the 5'-NT activity. Myricetin and quercetin were the most potent inhibitors for 5'-NT with IC50 values of 1.1 and 1.4 mu M, respectively. Kinetic analysis showed a mixed type of inhibitor for both myricetin (K-i = 1.5 mu M at pH 7.45), and quercetin (K-i = 0.6 mu M at pH 7.45). The K-m value for 5'-adenosine monophosphate (5-AMP) was determined with 77 mu M at pH 7.45. The differential inhibitory potencies of flavonoids seem to be structurally related (hydroxylation pattern). The results demonstrate that some flavonoids are strong inhibitors of 5'-NT activity which can be correlated to their pharmacological effects.