A new VP4 gene allele was identified by sequencing of two rotavirus strains recovered from infants with diarrhea. Strains HAL1166 (a VP7 serotype 8 human rotavirus from Finland) and PA169 (a VP7 serotype 6 human rotavirus from Italy) present 22-43% nucleotide sequence divergence and 14-45% amino acid divergence in the VP4 gene with rotaviruses belonging to various VP4 gene alleles previously reported. Cross-neutralization analysis between PA169 and rotaviruses belonging to the other recognized VP4 (P) serotypes had previously shown that none of them was similar to PA169. Hyperimmune serum prepared against a reassortant with the VP4 gene of strain PA169 and the VP7 gene of serotype 2 strain DS1 failed to neutralize previously recognized human rotavirus VP4 serotypes, indicating that PA169 represents a new VP4 serotype specificity. In addition, one of four neutralizing monoclonal antibodies directed to the VP4 of PA169 was specific for this virus, while three others had very limited cross-reactivity with rotaviruses possessing different VP4s.