Members of the p160 coactivator family (steroid receptor coactivator-1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1), and activator of thyroid and retinoic acid receptors (ACTR)) mediate transcriptional activation by nuclear receptors. After being recruited to the promoter by nuclear receptors, the p160 coactivator transmits the activating signal via two C-terminal activation domains, AD1 and AD2. AD1 is a binding site for the related coactivators cAMP-response element binding protein binding protein (CBP) and p300, whereas AD2 binds to another coactivator, coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase. The current study explored the cooperative functional and mechanistic relationships among GRIP1, CARM1, and p300 in transient transfection assays, where they enhanced the ability of the estrogen receptor (ER) to activate transcription of a reporter gene. The coactivator functions of p300 and CARM1 depended on the co-expression of GRIP1. Simultaneous co-expression of all three coactivators caused a synergistic enhancement of ER function. Deletion of the AD1 domain of GRIP1 abolished the ability of p300 to potentiate ER activity but had no effect on CARM1-mediated stimulation. In contrast, when the AD2 domain of GRIP1 was deleted, p300 still stimulated ER function through the mutant GRIP1, but CARM1 failed to do so. Thus, both binding of p300 to AD1 and binding of CARM1 to AD2 are required for their respective coactivator functions and for their synergy. Furthermore, CARM1 and p300 function independently through different activating domains of GRIP1, and their synergy suggests that they enhance transcription by different, complementary mechanisms.