Upon ligand binding the estrogen receptor alters its conformation, dimerizes, binds to estrogen response elements (EREs), recruits cofactors and initiates the formation of a transcriptional complex. In addition to estradiol binding, hormone receptor activity is modulated by phosphorylation at several key residues. Previous studies have shown that p21-activated kinase-1 (Pak1) and cyclic-AMP dependent protein kinase (PKA) can phosphorylate ERalpha at serine residue 305. However, the effects of serine 305 phosphorylation on ERalpha activity have not been fully characterized. To study these effects, ERalpha S305E and S305A mutants were created to mimic constitutively phosphorylated or un-phosphorylated states, respectively. Using yeast two-hybrid assays we showed that dimerization of ERalpha S305E was still ligand dependent. However, the capability of dimerization in the presence of estradiol was significantly higher in S305E compared to wild-type ERalpha. Transactivation assays demonstrated that phospho-mimetic ERalpha S305E is active in the absence of ligand. Chromatin immunoprecipitation (ChIP) analysis shows a change of in vivo DNA binding in which S305E mutant binds to ERalpha DNA target sequences and exhibits increased residency in the absence of ligand. We also observed increased cell growth in cells stably transfected with S305E ERalpha. Thus, we suggest that phosphorylation of S305 does not trigger ERalpha dimerization but increases binding to target gene promoters, which can lead to increased cell growth in the absence of estradiol. This implies a shift from hormone-induced activation of ERalpha to activation through phosphorylation, which could confer resistance to hormone based therapies for breast cancer.