Estrogen receptor (ER)alpha and ERbeta are localized in the nucleus and involved in the regulation of nuclear estrogen-responsive genes by 17beta estradiol (E2). In addition, recently others have shown that upon E2 binding, ERalpha localizes to the plasma membrane and initiates mitogen-activated protein kinase (MAPK)-mediated signal transduction. Previously, we reported that in liver, cultured rat hepatocytes and human HepG2 cells, estrogen treatment enhanced mitochondrial DNA (mtDNA)-encoded gene transcript levels. These effects were blocked by a specific antiestrogen, suggesting a role for the ER. Others have reported the presence of putative estrogen-responsive elements in mtDNA. These observations suggested the hypothesis that the ER localized in mitochondria and functioned directly to enhance the levels of mtDNA-encoded transcripts, analogous to what has been observed for the glucocorticoid hormone receptor. Using Western blot analysis, confocal immunofluorescence, immunogold electron microscopy, and gel electrophoresis mobility shift assays, we have demonstrated the estrogen-dependent presence of ERbeta and ERalpha within mitochondria of HepG2 and MCF-7 human breast tumor cells. Together, these results suggest that the ERs may act as transcription factors directly involved in the regulation by E2 of mtDNA transcription.