We report the cloning and characterization of a novel member of the Transcriptional Intermediary Factor 1 (TIF1) gene family, human TIF1γ. Similar to TIF1α and TIF1β, the structure of TIF1β is characterized by multiple domains: RING finger, B boxes, Coiled coil, PHD/TTC, and bromodomain. Although structurally related to TIF1α and TIF1β, TIF1γ presents several functional differences. In contrast to TIF1α, but like TIF1β, TIF1γ does not interact with nuclear receptors in yeast two-hybrid or GST pull-down assays and does not interfere with retinoic acid response in transfected mammalian cells. Whereas TIF1α and TIF1β were previously found to interact with the KRAB silencing domain of KOX1 and with the HP1α, MOD1 (HP1β) and MOD2 (HP1γ) heterochromatinic proteins, suggesting that they may participate in a complex involved in heterochromatin-induced gene repression, TIF1γ does not interact with either the KRAB domain of KOX1 or the HP1 proteins. Nevertheless, TIF1γ, like TIF1α and TIF1β, exhibits a strong silencing activity when tethered to a promoter. Since deletion of a novel motif unique to the three TIF1 proteins, called TIF1 signature sequence (TSS), abrogates transcriptional repression by TIF1γ, this motif likely participates in TIF1 dependent repression.