Although estrogen receptors (ERs) recognize 15-bp palindromic estrogen response elements (EREs) with maximal affinity in vitro, few near-consensus sequences have been characterized in estrogen target genes. Here we report the design of a genome-wide screen for high-affinity EREs and the identification of approximately 70000 motifs in the human and mouse genomes. EREs are enriched in regions proximal to the transcriptional start sites, and approximately 1% of elements appear conserved in the flanking regions (-10 kb to +5 kb) of orthologous human and mouse genes. Conserved and nonconserved elements were also found, often in multiple occurrences, in more than 230 estrogen-stimulated human genes previously identified from expression studies. In genes containing known EREs, we also identified additional distal elements, sometimes with higher in vitro binding affinity and/or better conservation between the species considered. Chromatin immunoprecipitation experiments in breast cancer cell lines indicate that most novel elements present in responsive genes bind ERalpha in vivo, including some EREs located up to approximately 10 kb from transcriptional start sites. Our results demonstrate that near-consensus EREs occur frequently in both genomes and that whereas chromatin structure likely modulates access to binding sites, far upstream elements can be evolutionarily conserved and bind ERs in vivo.