Estrogen signaling can occur through a nonclassical pathway involving the interaction of estrogen receptors (ER) with other transcription factors such as activator protein-1 (AP-1) and SP-1. However, there is little mechanistic understanding about this pathway, with conflicting results fromin vitro investigations. In this study, we applied the ChIP-on-chip approach to identify ERβ-binding sites on a genome-wide scale, identifying 1,457 high-confidence binding sites in ERβ-overexpressingMCF7 breast cancer cells. Genes containing ERβ-binding sites can be regulated by E2. Notably, ∼60% of the genomic regions bound by ERβ contained AP-1-like binding regions and estrogen response element-like sites, suggesting a functional association between AP-1 and ERβ signaling. Chromatin immunoprecipitation (ChIP) analysis confirmed the association of AP-1, which is composed of the oncogenic transcription factors c-Fos and c-Jun, to ERβ-bound DNA regions. Using a re-ChIP assay, we showed co-occupancy of ERβ and AP-1 on chromatin. Short interfering RNA-mediated knockdown of c-Fos or c-Jun expression decreased ERβ recruitment to chromatin, consistent with the role of AP-1 in mediating estrogen signaling in breast cancer cells. Additionally, ERα and ERβ recruitment to AP-1/ERβ target regions exhibited gene-dependent differences in response to antiestrogens. Together, our results broaden insights into ERβ DNA-binding at the genomic level by revealing crosstalk with the AP-1 transcription factor. ©2010 AACR.