p/CIP/SRC-3 is a member of a family of steroid receptor coactivators/nuclear receptor coactivators (SRC/NCoA) proteins that mediate the transcriptional effects of nuclear hormone receptors (NRs). Using deletion analysis we have mapped the location of two distinct activation domains in p/CIP (AD1 and AD2) capable of activating transcription in mammalian cells when fused to the Gal4-DNA binding domain. In addition to AD1 being coincident with the interaction domain for CBP, we demonstrate a novel in vivo interaction between the AD1 and GCN5. Overexpression of a Gal4-AD1 fusion protein in yeast leads to growth arrest that is relieved by mutation of genes encoding components of the SAGA complex including GCN5, ADA3, and SPT7. In addition, the AD1 of p/CIP and the ADA3 gene are shown to be essential for retinoic acid receptor alpha-dependent transcription in yeast. Transient transfection assays in mammalian cells indicate that GCN5 cooperates with p/CIP as a coactivator of RAR alpha-dependent transcription. Down-regulation of GCN5 using small interfering RNA in mammalian cells indicates that the AD1 domain and the RAR beta promoter activity are dependent, in part, on GCN5. Mutational analysis of AD1 has identified two helical motifs that are required for interactions with GCN5 and CBP. Taken together, these results support a model by which p/CIP functions as a ligand-dependent adapter, through specific protein-protein interactions with AD1, to recruit members from at least two distinct families of acetyltransferase proteins to NRs.