The p160 coactivators, steroid receptor coactivator-1 (SRC-1), transcriptional intermediary factor-2 (TIF2) and receptor-associated coactivator-3 (RAC3), as well as the coactivator/integrator CBP, mediate estrogen receptor-alpha (ERalpha)-dependent gene expression. Although these coactivators are widely expressed, ERalpha transcriptional activity is cell-type dependent. In this study, we investigated ERalpha interaction with p160 coactivators and CBP in HeLa and HepG2 cell lines. Basal and estradiol (E2)-dependent interactions between the ERalpha ligand-binding domain (LBD) and SRC-1, TIF2 or RAC3 were observed in HeLa and HepG2 cells. The extents of hormone-dependent interactions were similar and interactions between each of the p160 coactivators and the ERalpha LBD were not enhanced by 4-hydroxytamoxifen in either cell type. In contrast to the situation for p160 coactivators, E2-dependent interaction of the ERalpha LBD with CBP or p300 was detected in HeLa but not HepG2 cells by mammalian two-hybrid and coimmunoprecipitation assays, indicating that the cellular environment modulates ERalpha-CBP/p300 interaction. Furthermore, interactions between CBP and p160 coactivators are much more robust in HeLa than HepG2 cells suggesting that poor CBP-p160 interactions are insufficient to support ERalpha-CBP-p160 ternary complexes important for nuclear receptor-CBP interactions. Alterations in p160 coactivators or CBP expression between these two cell types did not account for differences in ERalpha-p160-CBP interactions. Taken together, these data revealed the influence of cellular environment on ERalpha-CBP/p300 interactions, as well as CBP-p160 coactivator binding, and suggest that these differences may contribute to the cell specificity of ERalpha-dependent gene expression.