Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. Intermediary factors interacting with their activation functions are required to mediate transcriptional stimulation. In search of such receptor interacting proteins, we have screened a human cDNA expression library and isolated a human protein that interacts in vitro with transcriptionally active estrogen receptors (ER). Sequence analysis reveals that this protein is the human homolog of mouse TIF1 (transcription intermediary factor 1) shown to enhance nuclear receptor ligand-dependent activation function 2 (AF2) in yeast. We have characterized the nuclear receptor binding site on hTIF1 and shown that a region of 26 residues is sufficient for hormone-dependent binding to the estrogen receptor. As shown by point mutagenesis, the AF2 activation domain of ER is required for the binding of hTIF1 but not sufficient, since a short region encompassing the conserved amphipathic alpha-helix corresponding to this domain fails to precipitate hTIF1. We also demonstrate that hTIF1 association with DNA-bound ER requires the presence of estradiol. Finally, we show that the interaction of hTIF1 with receptors is selective since strong in vitro hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.