Short heterodimer partner (SHP) is an orphan nuclear receptor that interacts with ER(alpha) and ERbeta and inhibits E2-induced transcription. We examined how SHP affects tamoxifen's estrogen agonist activity in endometrial cells. We report that SHP interacts with 4-hydroxytamoxifen (4-OHT) or E2-occupied ER(alpha) in a temperature-dependent manner in vitro. In transient transfection assays, SHP inhibited 4-OHT-stimulated reporter gene activity from an estrogen response element (ERE) in ER-positive RL95-2 but not in HEC-1A human endometrial carcinoma cells transfected with ER(alpha) or ERbeta. SHP inhibited E2-induced transcriptional activity in ER(alpha)- or ERbeta-transfected HEC-1A or Chinese hamster ovary-K1 cells. SHP inhibition of E2 activity was greater for ER(alpha) than ERbeta from the nonpalindromic ERE in the pS2 gene promoter in Chinese hamster ovary-K1 but not HEC-1A cells. Thus, ER subtype, cell type, and ERE sequence influence SHP repressor activity. An ER(alpha) mutant lacking activator function-1 showed reduced inhibition by SHP. In glutathione S-transferase pull-down experiments, SHP inhibited ER(alpha) dimerization, providing a possible mechanism to account for the inhibitory effect of SHP on ER activity. These results identify SHP as novel target for blocking 4-OHT agonist activity in endometrial cells.