Two monoclonal antibodies which exhibited neutralizing activity against the outer capsid VP7 of two or more rotavirus serotypes were utilized to locate amino acid residues involved in the formation of cross-reactive epitopes. Neutralizing monoclonal antibody (N-mAb) raised against the VP7 or porcine rotavirus Gottfried strain (VP7 serotype 4) neutralizes not only VP7 serotype 4 strains but also VP7 serotype 3, 6, 9, or 10 viruses. N-mAb raised against the VP7 of rhesus monkey rotavirus MMU 18006 strain (VP7 serotype 3) neutralizes serotype 3 viruses as well as porcine serotype 4 viruses. Neutralization-resistant variants were selected in the presence of these monoclonal antibodies. Analysis of nucleotide and amino acid (aa) changes in antigenic variants of serotype 3, 4, 6, 9, or 10 rotavirus selected in the presence of the serotype cross-reactive N-mAbs showed that: (1) in addition to variable regions VR-5 (aa 87-100) and VR-7 (aa 141-150), variable regions VR-8 (aa 208-224) and VR-9 (aa 235-242) are involved in cross-reactive neutralization; (2) the site of amino acid substitution on mutant VP7 selected by a single N-mAb can vary, resulting in mutants which exhibit antigenic differences; (3) an amino acid substitution can occur at the same position on the VP7 of different serotypes selected by a single N-mAb; or (4) the location of an amino acid substitution on the mutant VP7 selected by a single N-mAb can vary depending on the rotavirus serotype.