ARC92/ACID1 was identified as a novel specific target of the herpes simplex transactivator VP16 using an affinity purification procedure. Characterization of the protein revealed tight interactions with human Mediator mediated through a von Willebrand type A domain. ARC92/ACID1 further contains a novel activator-interacting domain (ACID), which it shares with at least one other human gene termed PTOV1/ACID2. The structure of ARC92/ACID1 is of ancient origin but is conserved in mammals and in selected higher eukaryotes. A subpopulation of Mediator is associated with ARC92/ACID1, which is specifically required for VP16 activation both in vitro and in mammalian cells, but is dispensable for other activators such as SP1. Despite many known targets of VP16, ARC92/ACID1 appears to impose a critical control on transcription activation by VP16 in mammalian cells.