A highly conserved composite regulatory element in the epithelial-specific enhancer of human papillomaviruses (HPVs) consists of an octamer motif separated by exactly 2 bp from a nonpalindromic NFI site. Point mutations within this composite element, created to prevent the binding of Oct-1 or NFI, result in up to 10- to 12-fold decrease in enhancer activity. A mutation preventing the binding of both proteins does not, however, result in any further decrease in activity suggesting a cooperative interaction between these two factors. Electrophoretic mobility shift assays provide evidence that the simultaneous binding of both factors to the composite element is indeed required for efficient activation. Furthermore, evidence demonstrating the inability of Oct-1 by itself to elicit a transcriptional response from this enhancer position suggests that Oct-1 does not activate transcription directly, but rather may play a crucial role in the viral enhancer by tethering NF1 to the composite element. This finding represents both a potentially important mechanism by which HPV gene expression can be regulated and an interesting model for the study of transcriptional cooperativity.