Two genotypes of Rubella virus have been described that differ by 8-9% at the nucleotide level in the E1 glycoprotein gene. Of these, genotype II (RGII) was only recently reported and in this study two RGII viruses, the BRDII vaccine strain and BR1 wild type strain, were characterized. Monoclonal antibodies against each of the virion proteins (capsid [C], glycoproteins E1 and E2) and polyclonal anti-rubella virus sera reacted similarly with purified virions from the RGII and reference RGI strains on Western gels, with the exception of one anti-E2 Mab, and thus the two genotypes are closely related antigenically. The genomic sequences of two genotype II (RGII) rubella virus strains were determined and compared with the six previously reported RGI sequences. The genomes of these viruses all contained 9762 nts and the lengths of the three untranslated regions (UTRs) and two open reading frames (ORF's) were identical. The overall difference between the RGI and RGII sequences at the nt level was approximately 8% and this difference was maintained across most of the genome. At the amino acid level, the RGI and RGII sequences differed overall by approximately 4%, however this difference was not uniform across the ORF's as the N-terminal third of P150 and the entirety of P90, both replicase proteins, were more conserved (<1% difference) while the C-terminal two thirds of P150 exhibited greater variation ( approximately 8% difference), including a hypervariable region between residues 771-801 within which divergence as great as 20-30% was detected. The parent wt virus of the BRDII vaccine was not available and its sequence was compared with the BR1 sequence to identify potential attenuating mutations. The BRDII and BR1 sequences varied at 252 residues (2.59%), including twelve in the UTRs and thirty coding differences in the ORF's. None of these differences in the BRDII sequence was vaccine-specific when compared with RGI wt and vaccine sequences and, therefore, there appeared to be no common pathway in the generation of live, attenuated rubella vaccines.