Analysis of estrogen receptor alpha and beta interplay with other transcription factors is critical to the understanding of how small molecules, the cognate ligands for these receptors, selectively regulate the mode and amplitude of gene transcription by affecting receptor activity. To better understand the molecular mechanisms of selective action of estrogen receptor ligands, we characterized estrogen receptor alpha and beta (ER) interaction with the p160 family of coactivators. We also investigated how these interactions are affected by binding of specific ligands. We show that ER alpha and beta utilize different LXXLL motifs for their interaction with p160 family members. We found that significant differences exist between the affinity of the nuclear receptor interacting domain (NRID) and interaction of separate LXXLL motifs with ERs. This result indicates that a single LXXLL motif is unlikely to be sufficient for interaction with receptors, and that regions other than LXXLL motifs also participate in ER-p160 complex formation. We found that ER alpha and beta have strong affinity preferences for particular coactivators. These results suggest that ER-mediated transcription is not driven by a random mixture of ER-coactivator complexes. We also show that some ER ligands are functionally specific. We describe a ligand that binds to both receptors, but enhances only ER beta interaction with SRC1 and SRC3 while exhibiting little effect on the ER alpha interaction with these proteins. Finally, we provide data that suggest how genistein may selectively recruit coactivators when liganded to ERs. It enhances the interaction of ERs with SRC1 and SRC3, but demonstrates a minimal effect on receptor interaction with DRIP205 and CBP.