The estrogen receptor-related receptor gamma (ERR gamma/ERR3/NR3B3) is the newest member of the ERR subfamily that also includes ERR alpha and ERR beta. All three isoforms share a high degree of amino acid identity especially in the DNA binding domain. ERR gamma is a constitutively active transcriptional activator that regulates reporter elements driven by steroidogenic factor 1 response element (SF-1RE) and estrogen response element. However, it has the highest potency on a derivative of SF-1RE present in the small heterodimer partner gene promoter called sft4 and unlike ERR alpha and -beta, it fails to activate a palindromic thyroid hormone response element. To investigate the mechanism behind this response element-specific differential transcriptional activity of ERR gamma, the interactions of ERR gamma and the aforementioned response elements was monitored. EMSA and chromatin immunoprecipitation assays demonstrated that ERR gamma binds to sft4, SF-1RE, and palindromic thyroid hormone response element albeit with different degrees of affinity, but causes hyperacetylation of sft4 and SF-1RE templates only. Limited proteolysis assays showed that ERR gamma, bound to different elements, shows differential trypsin sensitivity. A search for novel coregulators of ERR gamma led to the identification of receptor interacting protein 140 as a potent corepressor and peroxisome proliferator-activated receptor gamma coactivator 1 as a potent coactivator of ERR gamma. DNA-dependent pull-down and transient transfection assays demonstrated that, on different DNA elements, ERR gamma exhibits differential cofactor interactions, which in turn dictate its transcriptional activity. Because ERR gamma shows a similar tissue distribution as peroxisome proliferator-activated receptor gamma coactivator 1 and receptor interacting protein 140, these two coregulators may act as key components of ERR gamma-mediated transcription.