Transcription factors are known to interact with each other to modulate their transcriptional activity. In this study, we found that the transcriptional activity of human Erg (one of the Ets family-transcription factors) was repressed by several nuclear receptors, including human estrogen receptor ERalpha, nonsteroid receptors and orphan receptors. Conversely, Erg inhibited ERalpha-dependent transcription. These reciprocal functional interactions extended to other nuclear receptors such as thyroid hormone and retinoic acid receptors, as well as to Fli1, an ERG-related ETS factor. Although similarly inhibited by overexpression of the orphan nuclear receptors ERR1 and RORalpha, ERG activity was unaffected by either REV-ERBalpha1 or COUP-TFII. The antagonism between ERG and ERalpha did not depend on DNA binding inhibition or direct protein-protein interactions. Repression of ERalpha-dependent transcription required the carboxyterminal and aminoterminal transactivation domains of Erg whereas the carboxyterminal AF-2 domain of ERalpha was necessary for repression of Erg activity. Reciprocal inhibition between Erg and ERalpha was not alleviated by overexpressing CBP, SRC-1 or RIP 140, three nuclear coactivator proteins. A negative cross-talk observed between Erg and ERalpha expands their potential range of regulation and may be relevant in vivo, particularly in endothelial, urogenital and cartilaginous tissues where both factors are expressed.