Six neutralizing monoclonal antibodies (Mabs) and nine neutralization resistant viral variants (escape-mutant viruses (EMVs)) were used to further characterize the neutralization determinants of bluetongue virus serotype 10 (BTV10). The EMVs were produced by sequential passage of a highly cell culture adapted United States prototype strain of BTV10 in the presence of individual neutralizing Mabs. Mabs were characterized by neutralization and immune precipitation assays, and phenotypic properties of EMVs were characterized by neutralization assay. Sequencing of the gene segments encoding outer capsid proteins VP2 and VP5 identified mutations responsible for the altered phenotypic properties exhibited by individual EMVs. Amino acid substitutions in VP2 were responsible for neutralization resistance in most EMVs, whereas an amino acid substitution in VP5, without any change in VP2, was responsible for the neutralization resistance of one EMV. The data confirm that VP2 contains the major neutralization determinants of BTV, and that VP5 also can influence neutralization of the virus. The considerable plasticity of the neutralization determinants of BTV has significant implications for future development of non-replicating vaccines.