We analyzed cross-reactive neutralization epitopes on protein VP3 of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs), which showed a variety of interserotypic reactivity patterns when examined in a neutralization test and an enzyme-linked immunosorbent assay against 15 HRV and 2 animal RV strains. Serological study with the six cross-reactive N-MAbs revealed antigenic variations in some HRV strains within the same serotype as well as a marked antigenic difference between serotype 2 strains and serotype 1, 3, and 4 strains. Epitope analysis of the antigenic variants resistant to the six individual cross-reactive N-MAbs suggested the existence of at least three distinct cross-reactive neutralization epitopes on VP3 of HRV.