Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution. In one variant, a second nucleotide change occurred, but did not result in an amino acid change. Four independently selected 2F1 mutants showed mutations in four separate sites in antigenic regions A, C, and D. Two independently selected 1C10 variants had mutations in either the A region or an unreported site at amino acid 190. Two of the mutations resulted in the creation of new glycosylation sites which were utilized, but did not appear to greatly affect antigenicity. Of note, three of the mutants also demonstrated alterations in the migration patterns of gene 8 on PAGE. Such electrophoretic mobility shifts caused by single base neutralization escape mutations have not previously been reported for rotavirus. Since multiple mutations were selected with the same monoclonal antibody it appears that the antigenic regions of VP7, although widely separated in the linear sequence, are parts of a single, large and complex neutralization domain which includes amino acid 190 as well as the other previously reported epitopes.