Cross-reactive neutralization epitopes on VP4 of human rotavirus (HRV) were analyzed by the use of VP4-specific neutralizing monoclonal antibodies (N-MAbs) and MAb-resistant mutants. Seven anti-VP4 N-MAbs obtained in this study by using HRV serotypes 1 and 3 as immunizing antigens showed a variety of cross-reactivity patterns to 20 HRV strains with different serotype specificity in neutralization tests and a broader cross-reactivity to them was found for four N-MAbs in an enzyme-linked immunosorbent assay. On the basis of the reactivity patterns against rotaviruses in neutralization tests, these seven N-MAbs were classified into four groups. Cross-neutralization tests using a total of 12 pairs of MAbs and resistant mutants, including five pairs which had been prepared previously, showed that VP4 of HRV (strain KU) contained two independent antigenic regions. One, region C1, was recognized by a single MAb (YO-2C2) and the other was made up of two antigenic regions (C2 and C3) which overlapped operationally. Identification of amino acid substitution sites on VP4 of representative mutants of HRV strain KU indicated that amino acid positions 385 or 392 and 428 or 433 were critical for the C2 and C3 regions, respectively. These results suggested that regions C2 and C3 exist as conformational antigenic sites.