Estrogen receptor (ER)α and ERβ are transcription factors that can be activated by both ligand binding and growth factor signaling. Estradiol increases ER activity in part by enhancing interactions between its carboxy-terminal, ligand-binding domain (LBD) and the p160/SRC (steroid receptor coactivator) and p300/CBP (cAMP response element binding protein (CREB) binding protein) families of coactivators. In the absence of ligand and the LBD, these cofactors can also interact with the amino-terminal (A/B) domain of ERs in vitro. SRC-1 also enhances the ligand-independent activity of the full-length receptor. Both ligand-independent and estradiol-induced ER activity are increased by phosphorylation at specific serine (Ser) residues in the A/B domain (Ser^104/106 and Ser¹¹⁸ in ERα). In the case of ERβ, phosphorylation enhances the ligand-independent recruitment and action of SRC-1. We show here that unliganded ERα can activate endogenous gene expression in MCF-7 cells, and that this activation is mediated in part by a p160 coactivator. In transfected HeLa cells, we show that the full-length ERα can interact physically and functionally with p160/SRCs and CBP in the absence of ligand and that mutation of Ser^104/106/118 affects these interactions. Accordingly, ERα dephosphorylation decreases its ligand-independent interaction with SRC-1 and CBP in vitro. In HeLa cells, both Ser^104/106 and Ser¹¹⁸ impinge on SRC-1 action by two mechanisms: 1) a seemingly indirect effect on SRC-1 recruitment that requires other receptor domains in addition to the A/B, consistent with our finding that the ligand-independent interaction between the A/B and the LBD and its enhancement by SRC-1 depend in part on Ser^104/106/118; and 2) an effect on SRC-1 coactivation that can be observed in the absence of the LBD. Ser^104/106/118 can also affect coactivation by a subset of coactivators in the presence of hormone, albeit to a lesser extent than in its absence. Altogether, our observations suggest that the enhancement of ERα activity by p160/SRCs and CBP can be regulated by phosphorylation and stress the importance of using full-length receptors to assess the role of the activation function-1 in cofactor recruitment.