Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 27 human and animal rotavirus strains of serotype 3 in order to examine genetic variation within strains of identical serotype. Comparisons of the deduced amino acid sequences of the VP7s showed overall sequence identities of 85% or higher. A higher degree of overall VP7 sequence similarity was observed among strains from the same animal species when compared to strains from different animal species, suggesting that there are species-specific sequences in the VP7 protein. Alignment of the amino acid sequences demonstrated that amino acid sequence divergence among serotype 3 strains from different species was located primarily in previously established VP7 serotype-specific regions where genetic variation was identified among strains of different serotype. These regions were highly conserved among serotype 3 strains derived from the same species. The varying reactivities of three anti-VP7 monoclonal antibodies with the 27 strains was consistent with the occurrence of antigenic variation among serotype 3 strains. Moreover the reactivity of monoclonal antibodies correlated with the amino acid sequence found in two serotype-specific regions (VR5 and VR8). A computer-derived predicted phylogenetic tree suggests that rotavirus strains from different animal species belonging to serotype 3 are more closely related to each other than to rotavirus strains of different serotypes.