The ligand-occupied estrogen receptor alpha (ERalpha) initiates changes in gene expression through its interaction with target DNA. The capacity of ERalpha to modulate gene expression is influenced by the association of the receptor with a variety of coregulatory proteins. To further understand the role of these coregulatory proteins in ERalpha-mediated transcription, we have isolated and identified proteins associated with ERalpha when it is bound to the consensus estrogen response element. One of the proteins identified in this complex, flap endonuclease-1 (FEN-1), is required for DNA replication and repair. We show that FEN-1 interacts directly with ERalpha and enhances the interaction of ERalpha with estrogen response element-containing DNA. More importantly, chromatin immunoprecipitation and RNA interference assays demonstrate that endogenously expressed FEN-1 associates with the native pS2 gene in MCF-7 cells and influences estrogen-responsive gene expression. Interestingly, estrogen differentially regulates expression of FEN-1 in mouse uterine epithelial, stromal, and myometrial cells. Together, our studies help to elucidate the functional consequence of the ERalpha-FEN-1 interaction and increase our understanding of the elaborate regulatory mechanisms that drive estrogen-responsive gene expression and DNA repair.