Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand‐dependent activation function, AF‐2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF‐2 in yeast and interacts in a ligand‐dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF‐2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF‐2 antagonist hydroxytamoxifen cannot promote ER‐TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand‐dependent AF‐2. Interestingly, the TIF1 N‐terminal moiety is fused to B‐raf in the mouse oncoprotein T18.