The orphan receptor chicken ovalbumin upstream promoter transcription factor I (COUP-TF I) fully prevented not only the activation of the oxytocin gene by retinoic acid and thyroid hormone but also completely repressed the estrogen-dependent stimulation in transfected P19 EC cells. DNase I footprinting showed that the COUP-TF I protein bound to the 5'-flanking region of the oxytocin gene at the site of the distal composite hormone response element, which mediates the responses to estrogen, retinoic acid, and thyroid hormone. Electrophoretic mobility shift assay using this composite hormone response element as probe showed that COUP-TF I and the estrogen receptor competed for binding but did not form a heterodimer. The binding by COUP-TF I was stronger than the binding of the estrogen receptor. Thus, the mechanism of repression involves occupancy of integrated binding sites. By mutagenesis of the composite hormone response element, the COUP-TF I binding site and the estrogen response element could be separated, resulting in functional dissociation of the repressive action of COUP-TF I and the induction by estrogen. The results show that repression of gene expression by COUP-TF I is not limited to receptors that act through heterodimerization but also extends to the homodimer-forming estrogen receptor in a context-dependent manner. This interaction between COUP-TF I and the estrogen receptor may provide a physiological mechanism of selective antagonism of gene regulation by estrogens.