The neutralization antigens of human rotavirus VP7 were studied by producing eight neutralizing monoclonal antibodies to G type 9 rotaviruses F45 and WI61 and selecting antigenic variants resistant to neutralization by these monoclonal antibodies. Neutralization resistance patterns and sequence analysis of the antigenic variants indicated the presence of overlapping serotype-specific and serotype cross-reactive epitopes in antigenic region A, and one distinct type-specific epitope. Cross-reactive monoclonal antibodies were more tolerant of amino acid sequence change than type-specific monoclonal antibodies. The existence of a new antigenic region, F, including amino acids 235 to 242 was confirmed. This region contained a cross-reactive epitope not detectable in the presence of glycosylation at amino acid 238. This glycosylation also affected neutralization by a cross-reactive monoclonal antibody directed to antigenic region C. Antigenic regions A, B, C, and F all contain epitopes shared between G types, of which at least two (C and F) are affected by glycosylation. © 1993 Academic Press. All rights reserved.