The principal early promoter of human papillo-maviruses (HPVs), designated P97 in the case of HPV-16, contains four characteristically aligned cis-responsive elements, namely one binding site for Sp1, two for the viral E2 proteins, and the TATA box. The Sp1 binding site is needed to mediate activation of P97 by the remote epithelial-specific enhancer, and the two E2 binding sites contribute to a negative feedback-loop of viral gene expression. The Sp1 consensus motif and the TATA-box distal E2 binding site are spaced on all genital papillomaviruses by a single nucleotode. We show here that at physiological concentrations, the binding of E2 proteins and Sp1 are mutually exclusive events, since a bandshift analysis with nuclear extracts from ID13, a mouse cell line transformed by BPV-1, showed only the E2 or the Sp1 bandshift, but no complex indicative of the concomitant binding of both factors. Increasing concentrations of in vitro translated E2 protein compete efficiently with the Sp1 factor for binding to an oligonucleotide containing both binding sites. Interference between Sp1 and E2 protein binding is apparently relevant for P97 repression in vivo, since a mutational analysis revealed that both E2 binding sites are necessary for negative transcriptional regulation: Alone, neither the distal site, where E2 protein can induce Spi displacement, nor the proximal site, where E2 protein interferes with formation and function of the pre-initiation complex, have a significant effect, but two functional E2 binding sites lead to repression of P97.