The role of the carboxyl terminal F-domain of estrogen receptor (ERα) is uncertain, but evidence suggests that this region may impart internal restraint on ER dimerization in the presence of 17β-estradiol (E2). To identify the C-terminal residues affecting human ERα activation, we created a series of deletions and examined E2 induced receptor dimerization and transactivation. Deletion of the final 24 C-terminal amino acids of the F-domain (Δ7b) yielded a fivefold increase in dimerization, when compared to wild type (wt) ERα in the presence of 2nM E2, utilizing a yeast two-hybrid assay. This increase in dimerization is similar to that observed when the entire F-domain was deleted. Measurement of mutant:mutant homodimer formation yielded similar increases compared to mutant:wt interactions. Interestingly, a point mutation at the C-terminus (mut 3) showed increases in dimerization comparable to that of Δ7b in the presence of nanomolar amounts of E2. However, at sub-nanomolar levels of E2, mut 3 behaved similarly to wt ERα, whereas Δ7b maintained striking increases in dimerization. Determination of E2 binding affinity (Kd) constants revealed only marginal differences for wt and F-domain mutants, suggesting that the F-domain affects dimerization directly. We also observed enhanced interaction of F domain mutants with p160 family coactivator SRC1. Finally, transcriptional regulation of estrogen responsive reporters, 2XERE-LacZ and 3XERE-Luc in yeast and mammalian cells, respectively, reflected the increased propensity for dimerization by F domain mutants. Together, these data indicate that the C-terminal amino acids of ERα are critical for attenuation of E2 induced receptor dimerization and transcriptional activity.