The solution NMR structure of protein MED25(391-543), comprising the activator interacting domain (ACID) of subunit 25 of the human mediator, is presented along with the measurement of polypeptide backbone heteronuclear 15N-{1H} NOEs to identify fast internal motional modes. This domain interacts with the acidic transactivation domains of Herpes simplex type 1 (HSV-1) protein VP16 and the Varicella-zoster virus (VZV) major transactivator protein IE62, which initiate transcription of viral genes. The structure is similar to the β-barrel domains of the human protein Ku and the SPOC domain of human protein SHARP, and provides a starting point to understand the structural biology of initiation of HSV-1 and VZV gene activation. Homology models built for the two ACID domains of the prostate tumor overexpressed (PTOV1) protein using the structure of MED25(391-543) as a template suggest that differential biological activities of the ACID domains in MED25 and PTOV1 arise from modulation of quite similar protein-protein interactions by variable residues grouped around highly conserved charged surface areas.