Transcription of oncogenes E6 and E7 of human papillomavirus type 16 (HPV-16) from the P97 promoter is regulated by viral and cellular proteins. The transcription factor YY1 represses transcription through binding to cognate sequences in the long control region (LCR). In HPV-16 DNA from cervical carcinomas, mutations of YY1-binding sites have been identified that increase P97 activity 3-6-fold. A second, SP1-binding site has now been identified in the HPV-16 LCR (nt 7842-7847), which overlaps the YY1-binding site at positions 7840-7848. A point mutation within this YY1 site in viral DNA from a cervical cancer, previously shown to prevent YY1 binding, was shown to increase SP1 binding and P97 activity 4.7-fold. An engineered mutant eliminating SP1 binding showed only 1- to 1.6-fold increased P97 activity. It is concluded that competition between SP1 and YY1 for DNA binding plays a major role in YY1 repression mediated by the binding site at positions 7840-7848.