Tamoxifen (TAM) is successfully used for the treatment and prevention of breast cancer. However, many patients that are initially TAM responsive develop tumors that are antiestrogen/TAM resistant (TAM-R). The mechanism behind TAM resistance in estrogen receptor A (ERA)-positive tumors is not understood. The orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor (COUP-TF)-I interacts directly with 4-hydroxytamoxifen (4-OHT)-and estradiol (E 2 )-occupied ERA, corepressors NCoR and SMRT, and inhibit E 2 -induced gene transcription in breast cancer cells. Here we tested the hypothesis that reduced COUP-TFI and COUP-TFII correlate with TAM resistance. We report for the first time that COUP-TFII, but not COUP-TFI, is reduced in three antiestrogen/TAM-R cell lines derived from TAMsensitive (TAM-S) MCF-7 human breast cancer cells and in MDA-MB-231 cells compared with MCF-7. ERA and ERB protein expression was not different between TAM-S and TAM-R cells, but progesterone receptor (PR) was decreased in TAM-R cells. Further, E 2 increased COUP-TFII transcription in MCF-7, but not TAM-R, cells. Importantly, reexpression of COUP-TFII in TAM-S cells to levels comparable to those in MCF-7 was shown to increase 4-OHT-mediated growth inhibition and increased apoptosis. Conversely, knockdown of COUP-TFII in TAM-S MCF-7 cells blocked growth inhibitory activity and increased 4-OHT agonist activity. 4-OHT increased COUP-TFII-ERA interaction f2-fold in MCF-7 cells. COUP-TFII expression in TAM-R cells also inhibited 4-OHT-induced endogenous PR and pS2 mRNA expression. These data indicate that reduced COUP-TFII expression correlates with acquired TAM resistance in human breast cancer cell lines and that COUP-TFII plays a role in regulating the growth inhibitory activity of TAM in breast cancer cells.