An equinine rotavirus FI-14 strain, originally isolated from a diarrheic foal in New York state, was shown to belong to serotype 3 by neutralization assay. In addition, it was found to react with both subgroup I and subgroup II monoclonal antibodies by enzyme-linked immunosorbent assay (ELISA), thus representing the first rotavirus strain to exhibit both subgroup specificities. By using hybridoma technology, we successfully produced monoclonal antibodies directed against the major inner capsid protein VP6 (the sixth gene product) of FI-14 virus. Such monoclonal antibodies reacted specifically with either subgroup I or subgroup II rotaviruses thus demonstrating that the VP6 of FI-14 virus has both subgroup I- and subgroup II-specific epitopes. Four additional monoclones directed to the VP6 of FI-14 demonstrated distinct reactivities by ELISA with a panel of 49 rotavirus strains derived from 11 different animal and avian species. Thus, at least six distinct antigenic sites were shown to exist on VP6 of FI-14 virus. When these 49 rotavirus strains were arranged based on their reactivity patterns with the six representative monoclones, they fell into one of eight reactivity groups. Analysis of the reactivity patterns of rotaviruses derived from various animal species suggested that human rotaviruses may have two ancestral lineages: one (subgroup II, serotype 1, 3, and 4) with pig-human lineage, and the other (subgroup I, serotype 2) with bovine-simian-human lineage. When analyzed by radioimmunoprecipitation, the molecular weight of the FI-14 virus VP6 (subgroups I and II) appeared to be larger (approx 45K) than those (approx 42K) of rhesus monkey MMU18006 virus VP6 (subgroup I) or human Wa virus VP6 (subgroup II). By RNA-RNA hybridization analysis, the FI-14 virus was shown not to share significant homology with viruses belonging to the four known human rotavirus serotypes.