VP6, the most abundant protein of rotaviruses, contains epitopes that allow the classification of these viruses into four subgroups (SG), depending on the presence or absence of two epitopes called I and II. The subgroup-specific epitopes are conformational and appear to be present on trimeric but not monomeric VP6. We have identified on VP6 some of the amino acids that determine the reactivity of the subgroup-specific mAbs 255/60 and 631/9. A single amino acid mutation at positions 172 (Met to Ala) or 305 (Asn to Ala) was sufficient to change the subgroup specificity of the human rotavirus Wa VP6 protein from SGII to SGI/II, since either of these mutations allowed the protein to be recognized by the SGI mAb 255/60, while retaining its capacity to interact with the SGII mAb 631/9. In the case of the SGII epitope, the mutation of two contiguous amino acids (Ala305 Asn306 to Asn305 Ala306) in the porcine rotavirus YM VP6 protein (SGI) enabled the protein to be efficiently recognized by the SGII mAb 631/9, while causing the YM VP6 protein to lose its capacity to interact with mAb 255/60. These results suggest that both subgroup Abs interact with an antigenic domain in VP6 that is composed of at least two regions of the protein that, although distant in the linear sequence, might be in close proximity in the structured VP6 trimer.