Inhibition of estrone sulfatase activity offers the potential for breast cancer prevention therapy by blocking a route to estrogen synthesis. We have investigated the inhibition of this activity by natural flavonoids in a human hepatic microsomal preparation in vitro. The majority of studies were performed with a male liver, but male and female livers exhibited comparable estrone sulfatase activities. The natural flavonoids, quercetin, kaempferol, and naringenin, significantly inhibited estrone sulfatase activity with I 5 0 <10 μM for the most potent, quercetin. Estrone sulfatase activity in the liver microsomes was biphasic, with a high affinity, low capacity, low concentration activity (K m 14.3 μM, V m a x 0.5 nmol/min/mg protein), probably steroid sulfatase-catalysed, and a low affinity, high capacity, high concentration activity (K m 1.5 mM, V m a x 21.5 nmol/min/mg protein), probably arylsulfatase C or E-catalysed. The former activity was inhibited uncompetitively by quercetin, the latter competitively. Quercetin, a natural dietary constituent, is a potent inhibitor of estrone sulfatase in vitro, and thus has the potential to express antiestrogenic activity in vivo.