A series of estrone conjugates of the type estrone-3-O-C(O,S)-X have been prepared and evaluated for inhibition of human steroid sulfatase (STS). Among the carbamate (6), thiocarbamate (8), cyanate (7), formate (9), and acetate (10) analogs of estrone, only 9 was found to inhibit STS in a time- and concentration-dependent manner. With an IC(50) of 0.42 microM 9 is the first potent inactivator of STS which does not feature the sulfamate group. Furthermore a formate-type inhibitor featuring a benzoxazole moiety in place of the steroid skeleton (14) was prepared, suggesting a general principle of inactivation by the formate group. As the mode of action we propose an immediate transfer of the formyl moiety to a nucleophilic residue in the active site of STS.