Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.