A strong epithelial specific enhancer drives transcription of the human papillomavirus type 18 (HPV18) oncogenes. Its activity depends on the formation of a higher-order nucleoprotein complex (enhanceosome) involving the sequence-specific JunB/Fra2 transcription factor and the HMG-I (Y) architectural protein. Here we show that proteins from HeLa cell nuclear extract cover almost all of the HPV18 enhancer sequences and that it contains seven binding sites for the purified HMG-I (Y) protein, providing evidence for a tight nucleoprotein structure. Binding of HMG-I (Y) and the AP1 heterodimer from HeLa nuclear extract to overlapping sites of the core enhanceosome is cooperative. The integrity of this specific HMG-I (Y) binding site is as essential as the AP1 binding site for the enhancer function, indicating the fundamental role played by this architectural protein. We demonstrate that the CBP/p300 coactivator is recruited by the HPV18 enhanceosome and that it is limiting for transcriptional activation, since it is sequestered by the adenovirus E1A protein and by the JunB/Fra2 positive factor in excess. We show the involvement of JunB and p300 in vivo in the HPV18 transcription by chromatin immunoprecipitation of HPV18 sequences in HeLa cells.