Human papillomavirus (HPV) 8 belongs to the HPV types frequently associated with skin cancers of Epidermodysplasia verruciformis (EV)-patients. There are 33 nucleotides (M33 motif) in the 5'-part of the non-coding regulatory region of HPV8, which appear highly conserved among EV-specific HPVs and are consistently followed by an AP1 binding site. These sequences were shown to constitute an essential activator of transcription driven by the HPV8 late promoter P7535. The M33/AP1 element displayed properties of a constitutive enhancer, being also able to stimulate the activity of the heterologous thymidine kinase promoter in a position-independent manner. No protein binding could be detected within the 5'-part of the M33/AP1 region, which contributed significantly to the overall activity of the HPV8 enhancer. As shown by DNasel-footprinting, the central and the 3'-part of the enhancer region were involved in interactions with nuclear proteins. Three specific complexes could be observed in gel retardation tests with nuclear extracts from epithelial cells. One of these interactions involved the AP1 protein. Analysis of deletion and point mutations revealed binding of the AP1 protein to be essential for transcriptional activation, but DNA-protein interactions within M33 were important for maximal stimulation. The response to the phorbol ester TPA also required a cooperation of M33 and AP1.