The use of estrogen-based therapies and the selective estrogen receptor (ER) modulator (SERM), raloxifene, which are approved for postmenopausal osteoporosis, is associated with side effects such as uterine/breast hyperproliferation, thromboembolism, and hot flashes. A combination of a new SERM, bazedoxifene (BZA), and Premarin (conjugated estrogens; CE) is under investigation to mitigate the estrogen/SERM side effects with promising results in Phase III clinical trials. To explore the mechanism of BZA/CE action, we investigated the recruitment of cofactor peptides to ERalpha by components of CE and a mixture containing the 10 major components of CE with or without three different SERMs. Here, we demonstrate differential recruitment of cofactor peptides to ERalpha by the individual CE components using a multiplex nuclear receptor-cofactor peptide interaction assay. We show that estrone and equilin are partial agonists in comparison with 17beta-estradiol in recruiting cofactor peptides to ERalpha. Further, CE was more potent than 17beta-estradiol in mediating ERalpha interaction with cofactor peptides. Interestingly, BZA was less potent than other SERMs in antagonizing the CE-mediated cofactor peptide recruitment to ERalpha. Finally, in accordance with these biochemical findings, 17beta-estradiol and CE, as well as SERM/CE combinations, showed differential gene regulation patterns in MCF-7 cells. In addition, BZA showed antagonism of a unique set of CE-regulated genes and did not down-regulate the expression of a number of CE-regulated genes, the expression of which was effectively antagonized by the other two SERMs. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same beneficial effects that are observed in clinic by pairing BZA with CE.