The high mobility group protein HMG I(Y) and the transcription factor NF-kappa B are required for the activity of positive regulatory domain II (PRDII), a virus-inducible regulatory element of the human interferon-beta gene promoter. In this paper we provide evidence that HMG I(Y) is also required for the activity of PRDIV, a regulatory element that synergizes with PRDII. In this case, HMG I(Y) stimulates binding of activating transcription factor 2 (ATF-2) and the assembly of inducible complexes containing ATF-2 and c-Jun. Remarkably, HMG I(Y) also specifically interacts with the leucine zipper/basic region of ATF-2, and ATF-2 in turn interacts with NF-kappa B. We therefore propose that the HMG I(Y) plays a critical structural role in establishing transcriptional synergy between PRDII and PRDIV by promoting the activities and/or binding of NF-kappa B and ATF-2 and by facilitating their interaction.