Human papillomavirus type 16 (HPV-16) is the prototype strain among the malignant types of HPV in the western world. The main promoter, P97, located in front of the E6 ORF, has been shown to control expression of the oncogenes E6 and E7. These oncogenes are expressed continuously in HPV-16-transformed cells. In contrast to malignant HPV types, non-malignant HPV types have separate promoters driving the expression of E6 and E7. Experiments have shown that the translation of E7 is more efficient from monocistronic than bicistronic transcripts encoding both E6 and E7. Here, identification of a cluster of transcription start sites located in the E6 ORF of HPV-16 is presented. Transcripts from this region contain the E7 ORF as the first reading frame. The cluster consists of multiple transcription start sites located around nt 441. Additional transcription start sites were identified in a cluster around nt 480. A transcription start site has been identified previously at nt 480 but has never been characterized further. The region responsible for transcription activity was mapped to nt 272-448. Mutational analysis showed that initiation of transcription is independent of a TATA-box element, which is consistent with the finding of multiple transcription start sites. Furthermore, it is shown that proteins from HeLa and SiHa nuclear cell extracts bind to the two regions at nt 291-314 and 388-411, and that these two regions influence transcription activity in a cell type-dependent manner.