Insulin receptor substrate 1 (IRS-1) is a major signaling molecule activated by the insulin and insulin-like growth factor I receptors. Recent data obtained in different cell models suggested that in addition to its conventional role as a cytoplasmic signal transducer, IRS-1 has a function in the nuclear compartment. However, the role of nuclear IRS-1 in breast cancer has never been addressed. Here we report that in estrogen receptor alpha (ERalpha)-positive MCF-7 cells, (1) a fraction of IRS-1 was translocated to the nucleus upon 17-beta-estradiol (E2) treatment; (2) E2-dependent nuclear translocation of IRS-1 was blocked with the antiestrogen ICI 182,780; (3) nuclear IRS-1 colocalized and co-precipitated with ERalpha; (4) the IRS-1:ERalpha complex was recruited to the E2-sensitive pS2 gene promoter. Notably, IRS-1 interaction with the pS2 promoter did not occur in ERalpha-negative MDA-MB-231 cells, but was observed in MDA-MB-231 cells retransfected with ERalpha. Transcription reporter assays with E2-sensitive promoters suggested that the presence of IRS-1 inhibits ERalpha activity at estrogen-responsive element-containing DNA. In summary, our data suggested that nuclear IRS-1 interacts with ERalpha and that this interaction might influence ERalpha transcriptional activity.