The P670 promoter of HPV16 directs transcription of the virus late genes in the differentiating epithelium. We found that CCAAT/enhancer binding protein beta (C/EBPbeta), a key transcription factor that induces the terminal differentiation of keratinocytes, enhanced the P670-driven transcription in transient reporter assays in HeLa cells and human primary keratinocytes, whereas it inhibited, as reported previously, the transcription from the early P97 promoter. An electrophoretic mobility shift analysis identified two binding sites in the upstream region of P670 for a bacterially expressed C/EBPbeta. A chromatin immunoprecipitation analysis demonstrated that C/EBPbeta bound to these sites of the P670 reporter plasmid in HeLa cells. Nucleotide substitutions in these sites in the reporter plasmid abrogated the enhancement by C/EBPbeta in the transient HeLa and keratinocyte assays, indicating that the C/EBPbeta-binding to these sites is required for the enhancement of transcription from P670. These results suggest that C/EBPbeta is involved in enhancing transcription from the P670 during keratinocyte differentiation.