Tamoxifen is a selective estrogen receptor modulator widely used for the prophylactic treatment of breast cancer. In addition to the estrogen receptor (ER), tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS), which is involved in ERindependent effects of tamoxifen. In the present study, we investigate the modulation of the biosynthesis of cholesterol in tumor cell lines by AEBS ligands. As a consequence of the treatment with the antitumoral drugs tamoxifen or PBPE, a selective AEBS ligand, we show that tumor cells produced a significant concentration-and time-dependent accumulation of cholesterol precursors. Sterols have been purified by HPLC and gas chromatography, and their chemical structures determined by mass spectrometric analysis. The major metabolites identified were 5␣-cholest-8-en-3␤-ol for tamoxifen treatment and 5␣-cholest-8-en-3␤-ol and cholesta-5,7-dien-3␤-ol, for PBPE treatment, suggesting that these AEBS ligands affect at least two enzymatic steps: the 3␤-hydroxysterol-⌬ Tamoxifen is a selective estrogen receptor modulator (SERM) widely used for the treatment and the prevention of breast cancer (1). More than 20 years ago, Sutherland et al. (2) discovered that tamoxifen bound to a high affinity binding site that was different from the estrogen receptor (2). This site has been named the microsomal antiestrogen binding site (AEBS) 1 because it is localized in the microsomes of cells; it binds principally aryl aminoethoxy antiestrogens and has no affinity for estrogens (3). Two classes of selective ligands have been developed so far to selectively target the AEBS. The first class includes diphenylmethane derivatives of tamoxifen (DPPE) and N-pyrrolidino-2-[4-(benzyl)-phenoxy-ethanamine⅐HCl (PBPE) (4 -6), while the second class includes oxygenated derivatives of cholesterol such as 7-ketocholestanol (7-9). We and others (5, 10 -14) have shown that AEBS ligands inhibit the growth of tumor cell lines in vitro and in vivo, demonstrating that the AEBS was involved in the mediation of the effects of these structural classes of its cognate ligands. These compounds represent not only specific tools to study AEBS function but are also anticancer drug candidates because the selective AEBS ligand DPPE (Tesmilifene) was brought up to phase II and III clinical trials for the treatment of breast and prostate cancer in association with doxorubicin (15-17). Two principal points remained unsolved: the first is the precise molecular nature of the AEBS and the second the explanation of the difference observed between the nanomolar affinity of AEBS ligands and their micromolar effectiveness for growth control and cytotoxicity.We have been involved in the identification of the AEBS for several years. The AEBS can be found in most tissues in mammals and is abundant in microsomes of liver that contained 20 -30ϫ the amount found in tumor cell lines (18). For this reason, the liver have been chosen for the purification of the AEBS but the pharmacological profiles of the AEBS found in the ...