The nonpathogenic phenotype of the live rabies virus (RV) vaccine SPBNGAN is determined by an Arg-->Glu exchange at position 333 in the glycoprotein, designated GAN. We recently showed that after several passages of SPBNGAN in mice, an Asn-->Lys mutation arose at position 194 of GAN, resulting in GAK, which was associated with a reversion to the pathogenic phenotype. Because an RV vaccine candidate containing two GAN genes (SPBNGAN-GAN) exhibits increased immunogenicity in vivo compared to the single-GAN construct, we tested whether the presence of two GAN genes might also enhance the probability of reversion to pathogenicity. Comparison of SPBNGAN-GAN with RVs constructed to contain either both GAN and GAK genes (SPBNGAN-GAK and SPBNGAK-GAN) or two GAK genes (SPBNGAK-GAK) showed that while SPBNGAK-GAK was pathogenic, SPBNGAN-GAN and SPBNGAN-GAK were completely nonpathogenic and SPBNGAK-GAN showed strongly reduced pathogenicity. Analysis of genomic RV RNA in mouse brain tissue revealed significantly lower virus loads in SPBNGAN-GAK- and SPBNGAK-GAN-infected brains than those detected in SPBNGAK-GAK-infected brains, indicating the dominance of the nonpathogenic phenotype determined by GAN over the GAK-associated pathogenic phenotype. Virus production and viral RNA synthesis were markedly higher in SPBNGAN-, SPBNGAK-GAN-, and SPBNGAN-GAK-infected neuroblastoma cells than in the SPBNGAK- and SPBNGAK-GAK-infected counterparts, suggesting control of GAN dominance at the level of viral RNA synthesis. These data point to the lower risk of reversion to pathogenicity of a recombinant RV carrying two identical GAN genes compared to that of an RV carrying only a single GAN gene.