New World primates (NWP) exhibit a form of resistance to estrogens that is associated with overexpression of an estrogen response element (ERE) binding protein (ERE-BP) and an intracellular estradiol binding protein (IEBP). Both proteins suppress estradiol (E₂)-mediated transcription when over-expressed in estrogen receptor alpha (ER)^+VE cells. Whilst ERE-BP acts as a competitor for ERE occupancy by liganded ER, the function of IEBP and its human homologue, heat-shock protein 27 (hsp27), is less clear. In data presented here we have used estradiol (E₂)-responsive human MCF-7 breast cancer cells to show that IEBP/hsp27 can regulate estrogen signaling, as a cytosolic decoy for E₂ and as a protein chaperone for ER. Further co-immunoprecipitation, co-localization, yeast two-hybrid and GST-pull-down analyses indicate that IEBP/hsp27 also interacts with ERE-BP to form a dynamic complex which appears to cycle between the cytoplasm and nucleus during normal estrogen signaling. Over-expression of either IEBP/hsp27 or ERE-BP in MCF-7 cells resulted in abnormal subcellular distribution of the IEBP/hsp27 and ERE-BP, with concomitant dysregulation of ERE occupancy as determined by chromatin immunoprecipitation. We hypothesize that IEBP/hsp27 and ERE-BP not only cause hormone resistance in NWP but are also crucial to normal estrogen signaling in human cells. This appears to involve a physical association between the two proteins to form a complex which is able to interact with both E₂ and ER in cytosolic and nuclear compartments.