Human papillomavirus type-16 (HPV-16) is an epitheliotropic DNA tumor virus associated with the development of cervical carcinoma. The expression of HPV-16 early genes is driven by a cell-type-specific enhancer located in the long control region of the viral genome. We identified an element within the HPV-16 minimal enhancer that has enhancer activity and binds a nuclear factor, designated papillomavirus enhancer binding factor-1 (PEF-1). The element (called-FP-F by us and fp5e by Gloss et al. (EMBO J 6:3735-3743, 1987)) was originally identified as a footprint by DNase I protection experiments. The PEF-1 binding site is centered around a CCAAT box-like CCAAC element. Introduction of A-->T transversions into the CCAAC element of fp5e abolished binding of PEF-1 and concomitantly abolished enhancer function. fp5e resembles binding sites for the transcriptional activators CTF/NF-1 and AP-2; however, we showed that neither of these factors interacted with this element. PEF-1 has an apparent molecular weight of about 110 kDa, and we propose that it is a novel factor involved in the transcriptional activation of HPV-16 gene expression.