Steroid sulfatase regulates the formation of estrone from estrone sulfate (E1S) and dehydroepiandrosterone (DHA) from DHA sulfate. DHA can be converted to androstenediol, a steroid with potent estrogenic properties, and inhibition of steroid sulfatase activity is therefore an important therapeutic target. Because nonsteroidal steroid sulfatase inhibitors may offer some advantage for use in the treatment of breast cancer, 4-methylcoumarin-7-O-sulfamate (COUMATE) was synthesized and shown to be active in vitro. In this study, in vitro and in vivo techniques have been used to confirm that COUMATE, in contrast to the steroidal steroid sulfatase inhibitor estrone-3-O-sulfamate, is devoid of estrogenic activity. COUMATE did not stimulate the growth of MCF-7 breast cancer cells or uteri of ovariectomized rats, in contrast to estrone-3-O-sulfamate. COUMATE was orally active in vivo and after multiple dosing (10 mg/kg/day for 7 days) inhibited liver estrone sulfatase activity by 85%. Seven days after single or multiple dosing with COUMATE, liver estrone sulfatase activity was almost fully restored. Measurement of estrone sulfatase activity in WBCs revealed a degree of inhibition similar to that detected in liver samples. COUMATE was able to completely block the ability of E1S to stimulate uterine growth in ovariectomized rats. The development of a potent nonsteroidal, nonestrogenic steroid sulfatase inhibitor should allow the therapeutic potential of this type of therapy to be evaluated.