Nuclear receptor (NR)-mediated transcription is driven by dynamic multiprotein coactivator complexes, the composition of which is thought to determine the biological activity of NRs at specific promoters. The extent to which NRs discriminate between a spectrum of potential binding partners is intuitively a function of the inherent affinities of these individual interactions. Using real time interaction analysis with BIAcore, we evaluated the affinities and kinetics of the interactions of full-length members of the SRC/p160 coactivator family with estrogen receptor alpha (ER alpha) and ER beta bound to a variety of ligands. We substantiate that 17beta-estradiol enhances the affinity of ER-SRC/p160 interactions, whereas 4(OH)-tamoxifen, raloxifene, and ICI-182,780 inhibit these interactions. We show that a well defined, ER isoform-specific hierarchy governs the association of liganded ERs with full length SRC/p160 family members. Moreover, our data indicate that the interaction affinities of the full-length SRC/p160s with ERs are significantly higher then those of the NR interaction domains of the same coactivators, indicating that portions of coactivator molecules distinct from NR interaction domains might participate in receptor-coactivator complex formation. Finally, the interaction kinetics of SRC/p160s with ERs are consistent with a bipartite model, involving initial rapid formation of an unstable intermediate complex, and a subsequent slower reaction leading to its stabilization. We interpret our results as evidence that hierarchical coactivator interaction affinities are an important source of diversity in NR-mediated signaling and that the complexity of receptor-coactivator cross-talk might be best understood in the context of full-length molecules.