Transcription of the transforming genes E6 and E7 of the human papillomavirus type 18 (HPV18) can be repressed by the product of the E2 open reading frame. Mutations were introduced in the cis-responsive elements upstream from the E6 and E7 transcriptional promoter, P105. The effect of these mutations in the absence or presence of E2 was examined in the human cervical carcinoma cell line C33, transfected with expression plasmids. In the presence of HPV18 E2, repression was relieved only when both of the two E2 binding motifs, immediately proximal to the P105 TATA box, were mutated. Mutation of a third E2 binding site, 110 nucleotides upstream of the TATA box, led to a slight but consistent activation in the presence of the full-length E2. Therefore, three E2 binding sites appear to be involved in the negative regulation of the P105 promoter by HPV18 E2. In the presence of the BPV1 E2 protein, only the E2 binding site most proximal to the promoter TATA box was involved in the P105 repression. The comparative analysis of the in vitro and in vivo properties of the human and bovine E2 proteins on the HPV18 P105 promoter provides new insights into the mechanism of transcriptional repression, which appears to involve steric hindrance at the site of transcriptional initiation.