Mutants of enterovirus type 71 with respect to plaque size were selected from two American and two Japanese strains, and the antigenic relationship between the mutants was analyzed using immune animal and patients' sera. All four strains were mixtures of m+ (big) and m (minute) plaque mutants that were serologically different from each other. Thus, it appears that selected m+ and m mutants must be used for identification and antigenic analysis of isolates of enterovirus type 71. In patients with hand, foot, and mouth disease, the antibody responses to the m mutants of all four strains were significant, but responses to the m+ mutants were poor or absent. No antibody was detected if the current strain including the m+ mutant was used. Thus, the selected m mutant should be used for serologic diagnosis of infection with enterovirus type 71.