Monoclonal antibodies (MAbs) raised against a synthetic peptide including residues 156-170 of protein VP2 of human rhinovirus type 2 (HRV2) have previously been shown to be of differing specificities. The basis for these differences has now been examined in greater detail by ELISA, radioimmunoprecipitation and virus neutralization. Reactions with a panel of HRV2 mutant viruses indicated that substitution of some residues could enhance the apparent activity of one of the neutralizing anti-peptide MAbs. For one such substitution, VP2 P164H, there appeared to be a correlation between increased neutralizing activity and enhanced binding. Mapping experiments identified two overlapping neutralization epitopes (amino acids 156-163 and 160-165) and several non-neutralizing epitopes. Although some differences in antibody reactivity were due to epitope specificity alone, the explanation for others was less obvious. Significantly, the majority of MAbs that recognized, and in some cases neutralized, native virus had the same minimum binding sequence and critical residue requirement as others which recognized virus particles only after distortion. This demonstrates that factors other than the linear sequence of the peptide can be crucial in determining the fine specificity, and hence biological relevance, of peptide antigens.