A significant frequency of amino acid substitutions, that affect important antigenic sites on capsid protein VP1, has been found among viral preparations used for the production and for challenge, in protection assays, of foot-and-mouth disease (FMD) vaccines. The amino acid substitutions present in one of the viruses studied abolished its reactivity with two neutralizing monoclonal antibodies that recognized different epitopes on VP1. Thus, the results obtained illustrate the high potential for antigenic variation introduced by the multiple cycles of growth usually undergone by the strains used for the production and challenge of FMD vaccines.