Transcriptional activation of the virus inducible enhancer of the human interferon-β (IFN-β) gene in response to virus infection requires the assembly of an enhanceosome, consisting of the transcriptional activators NF-κB, ATF-2/c-Jun, IRFs and the architectural protein of the mammalian high mobility group I(Y) [HMG I(Y)]. Here, we demonstrate that the first step in enhanceosome assembly, i.e. HMG I(Y)-dependent recruitment of NF-κB and ATF-2/c-Jun to the enhancer, is facilitated by discrete regions of HMG I and is mediated by allosteric changes induced in the DNA by HMG I(Y) and not by protein–protein interactions between HMG I(Y) and these proteins. However, we show that completion of the enhanceosome assembly process requires protein–protein interactions between HMG I(Y) and the activators. Finally, we demonstrate that once assembled, the IFN-β enhanceosome is an unusually stable nucleoprotein structure that can activate transcription at high levels by promoting multiple rounds of reinitiation of transcription.