Abstract To advance understanding of mechanisms leading to biological and transcriptional endpoints related to estrogen action in the mouse uterus, we have mapped ERα and RNA polymerase II (PolII) binding sites using chromatin immunoprecipitation followed by sequencing of enriched chromatin fragments. In the absence of hormone, 5184 ERα-binding sites were apparent in the vehicle-treated ovariectomized uterine chromatin, whereas 17,240 were seen 1 h after estradiol (E2) treatment, indicating that some sites are occupied by unliganded ERα, and that ERα binding is increased by E2. Approximately 15% of the uterine ERα-binding sites were adjacent to (<10 kb) annotated transcription start sites, and many sites are found within genes or are found more than 100 kb distal from mapped genes; however, the density (sites per base pair) of ERα-binding sites is significantly greater adjacent to promoters. An increase in quantity of sites but no significant positional differences were seen between vehicle and E2-treated samples in the overall locations of ERα-binding sites either distal from, adjacent to, or within genes. Analysis of the PolII data revealed the presence of poised promoter-proximal PolII on some highly up-regulated genes. Additionally, corecruitment of PolII and ERα to some distal enhancer regions was observed. A de novo motif analysis of sequences in the ERα-bound chromatin confirmed that estrogen response elements were significantly enriched. Interestingly, in areas of ERα binding without predicted estrogen response element motifs, homeodomain transcription factor-binding motifs were significantly enriched. The integration of the ERα- and PolII-binding sites from our uterine sequencing of enriched chromatin fragments data with transcriptional responses revealed in our uterine microarrays has the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine and other estrogen target tissues.