Human papillomavirus (HPV) 16 is highly associated with premalignant and malignant anogenital epithelial lesions. The transforming function resides within the viral E7 open reading frame protein. We have defined three immunodominant linear B-epitopes in the E7 protein. In the present study, we determine the contribution of individual amino acid residues to antibody binding of these three epitopes using replacement set analysis. In this approach, each epitope residue is substituted in turn by each of the other 19 genetically encoded amino acids to produce analogues which are tested for specific monoclonal antibody binding. We demonstrate the specificity of the monoclonal antibodies for epitopes of HPV 16 E7 in binding studies using synthetic epitope analogues of other HPV genotypes. Comparison between HPV 16 and other HPV genotypes suggests that variability in amino acid composition at the E7 epitopic sites does not appear to be host-antibody driven.