We have reported previously that a 636-bp fragment spanning the 5' two-thirds of the human papillomavirus type 6 (HPV6)-W50 long control region (LCR) functions as a transcriptional silencer (A. Farr, S. Pattison, B.-S. Youn, and A. Roman, J. Gen. Virol. 76:827-835, 1995). We have utilized nested deletion analyses to implicate a 66-bp sequence which appears to be critical for this activity. A comparison of the transcriptional regulatory activities of the LCRs of HPV6-W50 and HPV6b (which has a 94-bp deletion, resulting in the elimination of the 66-bp sequence) indicates that sequences within the 94-bp region negatively regulate the activity of the intact HPV6 LCR. Two sequence-specific DNA-protein interactions were visualized via electrophoretic mobility shift assays. One of the binding events is mediated by the transcriptional repressor CCAAT displacement protein (CDP), a factor which is active in undifferentiated cells but inactive in terminally differentiated cells. This conclusion is based on the following three lines of evidence: (i) a consensus CDP binding site oligonucleotide serves as a competitor in band shift assays, (ii) the band shift complex is not seen when a CDP-negative nuclear extract is used, and (iii) anti-CDP antiserum specifically inhibits the binding. These studies identify a DNA-protein interaction occurring within the 5' end of the LCR which may be important in maintaining the tight link between keratinocyte differentiation and HPV gene expression.