The function of the 5' region of the upstream regulatory region (URR) in regulating E6/E7 expression in cancer-associated papillomaviruses has been largely uncharacterized. In this study we used linker-scanning mutational analysis to identify potential cis regulatory elements contained within a portion of the 5' region of the URR that are involved in regulating transcription of the E6/E7 promoter at different stages of the viral life cycle. The mutational analysis illustrated differences in the transcriptional utilization of specific regions of the URR depending on the stage of the viral life cycle. This study identified (i) viral cis elements that regulate transcription in the presence and absence of any viral gene products or viral DNA replication, (ii) the role of host tissue differentiation in viral transcriptional regulation, and (iii) cis regulatory regions that are effected by induction of the protein kinase C pathway. Our studies have provided an extensive map of functional elements in the 5' region (nuncleotides 7259 to 7510) of the human papillomavirus type 31 URR that are involved in the regulation of p99 promoter activity at different stages of the viral life cycle.