Coactivators are required for activation of target genes by nuclear receptors. A well-studied class of coactivators, the p160 proteins, use short nuclear receptor interaction domains (NR boxes) to bind to the activated ligand-binding domain of a nuclear receptor. To investigate how selective estrogen receptor modulators (SERMs) affect NR box recruitment, we compared the recruitment of p160 NR box peptides to the estrogen receptor (ER)alpha and ER beta in the presence of 17beta-estradiol (E2), 4-OH tamoxifen (4-OH Tam), LY 117018 (a raloxifene analog), and ICI 182780 (ICI, an ER antagonist). Our coactivator interaction assay utilizes time-resolved fluorescence technology to assess the binding of the 10 NR boxes derived from the three known p160 coactivators (SRC-1, -2, -3) to the ER subtypes in the presence of each ligand. The SERMs we studied did not increase NR box binding to either ER alpha or ER beta, but instead were potent antagonists decreasing estradiol-dependent NR box binding. We also demonstrated inverse agonism for all of the SERMs tested as they dose-dependently decreased hormone-independent NR box binding to ER beta. Therefore, the SERMs studied behave as antagonists of ER alpha and ER beta NR box binding and do not increase coactivator NR box binding to either ER subtype. In addition, we examined the preference of E2-bound ER alpha and ER beta for various naturally occurring NR boxes including the 10 SRC boxes as well as the motifs from PGC-1, TRBP, TRAP220, and CBP. Interestingly, a clear preferential pattern of interaction was noted that was receptor specific.