Transcription of human papillomaviruses (HPV) is regulated by enhancer sequences located in the upstream regulatory region. The factors regulating expression of one of the high risk genital HPV types, HPV 31b, were investigated using transient expression and protein binding assays. A region of 262 base pairs in length was identified as the minimal functional enhancer and a series of five protected binding sites were observed by footprint analyses. Electrophoretic mobility shift assays demonstrated that AP1, Oct-1, as well as three novel factors bound these sequences. Mutational analyses indicated that AP1 synergistically activated the HPV31b enhancer together with either of two novel factors. One of these novel factors bound a sequence similar to an NF1 site but was distinct from NF-1. The second factor bound sequences bearing similarity to KRF-1 binding sites which have previously been characterized in HPV 18. Competition binding assays demonstrated that this factor was not identical to KRF-1. Additional studies implicated Oct-1 as a negative regulator of HPV 31b expression as mutation of Oct-1 binding sequences resulted in an increase in viral expression. None of the factors observed to be important for HPV 31b enhancer activity was found exclusively in epithelial cells and instead were detected in a variety of cell types. Of these factors, AP1 binding correlated most strongly with enhancer function in a variety of cell types, implicating it as a principal regulator of HPV expression. Variations in the constituents of the AP1 complex that bind the HPV 31b enhancer were also observed in different cell types, suggesting that changes in the distribution of jun proteins may play a significant role in determining the tropism of HPV. These results indicate that AP1 may be a common regulator for various HPV types and that it contributes to enhancer specificity. In addition, a set of novel factors, which may be specific for each HPV type, act synergistically with AP1 for full activation of the enhancer.