We present evidence that transcriptional activation of the human interferon-beta (IFN beta) gene requires the assembly of a higher order transcription enhancer complex (enhanceosome). This multicomponent complex includes at least three distinct transcription factors and the high mobility group protein HMG I(Y). Both the in vitro assembly and in vivo transcriptional activity of this complex require a precise helical relationship between individual transcription factor-binding sites. In addition, HMG I(Y), which binds specifically to three sites within the enhancer, promotes cooperative binding of transcriptional factors in vitro and is required for transcriptional synergy between these factors in vivo. Thus, HMG I(Y) plays an essential role in the assembly and function of the IFN beta gene enhanceosome.