Two new human rotavirus (HRV) strains, PA151 and PA169, with subgroup I specificity and a long RNA pattern, yet with a serotype G (VP7) specificity different from those of any of the six well-established HRV serotypes (G1 to G4, G8, and G9), were isolated 3 months apart from two children with acute gastroenteritis in Sicily, southern Italy, in the winter season of 1987 and 1988. The HRV isolates were adapted to growth in cell cultures and were then characterized by neutralization and RNA-RNA (Northern blot) hybridization. Cross-neutralization studies with type-specific immune sera to RV serotypes 1 to 10 showed the antigenic relatedness of the two strains with serotype 6 bovine strains UK and NCDV. Monoclonal antibodies to VP7 of UK were able to recognize UK and NCDV strains as well as both HRV isolates. Cross-hybridization studies showed a genetic relatedness of PA151 and PA169 to bovine strains for all genes except gene 4. Gene 4 of PA151 appeared to be genetically related to that of AU228 (a human strain of subgroup I and with serotype G3 specificity that belongs to a feline genogroup), whereas gene 4 of PA169 appeared to be unique, yet it was related to gene 4 of two recently reported subgroup I HRV strains, one (PA710) with serotype G3 specificity and the other (HAL1271) with serotype G8 specificity. The new HRV strains must be taken into consideration when deciding strategies for the development of an effective RV vaccine.