Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ERalpha and ERbeta appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ERalpha and ERbeta. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ERbeta preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.