Fixed rabies virus strains (ERA and CVS) produce a fatal paralytic disease in mice after intracerebral or intramuscular injection. Some antigenic mutants of both CVS and ERA viruses with a substitution in position 333 of the glycoprotein (arginine is replaced either by a glutamine, a glycine, or an isoleucine) are totally avirulent for adult mice whatever the dose and the route of inoculation. Here we report an exhaustive investigation of the effect of amino acid 333 on viral virulence. New antigenic mutants were isolated from either CVS, CVS derivatives, or SADBern having arginine in position 333 encoded by CGG, AGG, CGU, or AGA respectively. This study shows that when arginine is replaced by either a leucine, an isoleucine, a methionine, a cysteine, or a serine, the antigenic mutant is also totally avirulent. But when arginine is replaced by a lysine it is still pathogenic although the LD50 by the intracerebral route is higher. Furthermore 41 independent virulent revertants were isolated from four avirulent mutants (with a glycine, a glutamine, a methionine, or a serine in position 333 of the glycoprotein). Thirty-nine regained an arginine at position 333 and 2 had a lysine. From this analysis it appears that the presence of a positively charged amino acid (arginine or lysine) in position 333 of the glycoprotein is necessary for viral virulence.