Most studies on the regulation of gene expression in human papillomaviruses (HPV) have focused on the promoter for the early genes E6 and E7. This promoter is located at the junction between the long control region and the E6 open reading frame. RNA mapping studies have suggested that additional promoters may exist in other parts of the genome. In this study, we used a combination of transcription in vitro and an analysis of RNA produced in vivo in transfected cells to identify three novel promoters in the genome of human papillomavirus type 18. These promoters are located in front of the E2 gene (P2598), within the E2 coding sequences (P3036), and at the end of the L2 open reading frame (P5600). They were active in HeLa cells, as shown by a chloramphenicol acetyltransferase assay. The activity of the P3036 promoter was stimulated by the bovine papillomavirus type 1 E2 protein.