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 microM, respectively. Kinetic analysis showed a mixed type of inhibitor for both myricetin (Ki = 1.5 microM at pH 7.45), and quercetin (Ki = 0.6 microM at pH 7.45). The K(m) value for 5'-adenosine monophosphate (5-AMP) was determined with 77 microM 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.