Estrogen receptors (ER) regulate gene transcription by interacting with regulatory elements. Most information regarding how ER activates genes has come from studies using a small set of target genes or simple consensus sequences such as ERE, AP-1 and Sp1 elements. However, these elements cannot explain the differences in gene regulation patterns and clinical effects observed with E₂ and SERMs. To obtain a greater understanding of how E₂ and SERMs differentially regulate genes it is necessary to investigate their action on a more comprehensive set of native regulatory elements derived from ER target genes. Here we used chromatin immunoprecipitation-cloning and sequencing (ChIP-CS) to isolate 173 regulatory elements associated with ER. Most elements were found in the introns (38%) and regions greater than 10 kB upstream of the transcription intiation site (38%). 24% of the elements were found in the proximal promoter region (<10 kB). Only 11% of the elements contained a classical ERE. 23% of the elements did not have any known response elements, including one derived from the naked cuticle homolog gene, which was associated with the recruitment of p160 coactivators. Transfection studies found that 80% of the 173 elements were regulated by E₂, raloxifene or tamoxifen with ER or ER. Tamoxifen was more effective than raloxifene at activating the elements with ER, whereas raloxifene was superior with ER. Our findings demonstrate that E₂, tamoxifen and raloxifene differentially regulate native ER regulatory elements isolated by ChIP with ER and ER.