A single promoter has so far been found in the long control region (LCRs) of human papillomavirus-16 (HPV-16). Multiple promoters exist in the LCRs of several other papillomaviruses, which are spliced to become mRNAs for late and some early genes. Here we have investigated whether such promoters exist in the LCR of HPV-16. In in vitro transcription experiments, we detected a strong transcript starting 280 bp downstream from the 3′ end of the L1 gene between a nuclear matrix attachment region and the epithelial-specific enhancer. Promoter activity coincides with a GCCATTTT motif, which binds the transcription factor YY1 (YY1-7436). The A of this motif is the first nucleotide of the transcripts and identifies YY1-7436 as an initiator. Genomic segments with YY1-7436 initiate expression of a luciferase reporter gene in transfection experiments. Mutational analysis of YY1-7436 suggests, however, that promoter function originates from another factor but YY1, which can contact overlapping sequences. Promoter activity of YY1-7436 is modulated by upstream A-T-rich sequences, which bind the basal transcription factor TFIID, and it is stimulated by the viral E2 protein binding to a downstream E2 binding site. In differentiating W12 cells, which contain episomal HPV-16 copies, we detected transcripts including LCR sequences downstream of YY1-7436, which were differentially spliced to early and late genes. However, we could not detect 5′ ends mapping to YY1-7436, but we detected two novel HPV-16 promoters within the L1 gene. Conservation of the arrangement of the YY1 and E2 binding sites suggests a role in important biological functions, which, however, is difficult to confirm in every type of cell culture. The study of W12 cells complements the examination of YY1-7436 and points to yet undetected promoters upstream of the LCR.