We present evidence that transcriptional activation of the human interferon-p (IFNP) 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 l (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 l (Y), which binds specifically to three sites within the enhancer, promotes cooperative binding of transcription factors in vitro and is required for transcriptional synergy between these factors in vivo. Thus, HMG l (Y) plays an essential role in the assembly and function of the IFNp gene enhanceosome.