Monoclonal antibodies have been produced and used to map the functional topography of the surface proteins of rhesus rotavirus (RRV) that mediate viral neutralization. Ten monoclonal antibodies directed to VP7 were studied in neutralization assays and competitive binding studies. A large neutralization domain with several interrelated epitopes on VP7 was apparent. Twelve monoclonal antibodies directed to VP3 were used in similar studies and delineated at least 2 distinct neutralization domains on that protein. Neutralizing monoclonal antibodies directed at both VP3 and VP7 were used to isolate viral antigenic variants, which were then studied in neutralization and hemagglutination inhibition assays. The viral variant studies, while confirming the general conclusions obtained from the competitive binding studies, allowed the apparent distinction of two separate neutralization domains on VP7 and three on VP3. All VP7-specific monoclonal antibodies (mAb) mediated serotype-specific neutralization, but a VP3-specific mAb was identified that neutralized rotaviruses of three distinct serotypes. No alteration of viral virulence was apparent in studies of suckling mice orally inoculated with antigenic variant viruses selected with our panel of neutralizing VP3 or VP7-specific mAbs.