In benign squamous lesions and in organotypic epithelial cultures, the human papillomavirus (HPV) E6 and E7 genes are transcriptionally up-regulated in differentiated, spinous keratinocytes. We previously identified sequence elements in the enhancer-promoter regions of HPV types 18 and 11 important for this promoter regulation by using the bacterial LacZ reporter gene in stratified raft cultures of primary human keratinocytes (PHKs) or in submerged, proliferating cultures acutely transduced with recombinant retroviruses. Notably, mutations in the promoter-proximal Sp1, Oct1, and AP1 sites each significantly reduce reporter activity in differentiated cells, indicating that the bound factors are transcription transactivators. In the present study, we performed further mutagenesis on distal motifs in the HPV- 11 regulatory region in PHKs in submerged and raft cultures. Mutations in an AP2-like site, three individual NF-1 sites, or five NF-1 sites collectively reduced promoter activity slightly in differentiated cells. A mutation in a putative glucocorticoid response element had no discernable effect in the presence or the absence of dexamethasone. However, mutations in a C/EBP binding site, especially the distal site, strikingly up-regulated reporter gene expression, particularly in basal and lower spinous cells, implicating bound protein as a transcription repressor. Collectively, these results demonstrate that the overall differentiation-dependent papillomaviral gene expression observed in vivo and in vitro involves promoter repression in the lower strata and activation in the upper, differentiated strata.