An array of regulatory protein and multi-subunit cofactors has been identified that directs eukaryotic gene transcription. However, establishing the specific functions of various related cofactors has been difficult owing to the limitations inherent in assaying transcription in animals and cells indirectly. Here we describe, using an integrated chromatin-dependent reconstituted transcription reaction, the purification and identification of a multi-subunit cofactor (PBAF) that is necessary for ligand-dependent transactivation by nuclear hormone receptors. A highly related cofactor, human SWI/SNF, and the ISWI-containing chromatin-remodelling complex ACF both fail to potentiate transcription. We also show that transcriptional activation mediated by nuclear hormone receptors requires TATA-binding protein (TBP)-associated factors (TAFs) as well as the multi-subunit cofactors ARC/CRSP. These studies demonstrate functional selectivity amongst highly related complexes involved in gene regulation and help define a more complete set of factors and cofactors required to activate transcription.