Antigenic variants of CVS-11 strain of rabies virus were selected after treatment of virus populations with monoclonal antibodies directed against the glycoprotein antigen of the virus. These variants resisted neutralization by the hybridoma antibody used for their selection. Two independently mutating antigenic sites could be distinguished when five variants were tested with nine hybridoma antibodies. The frequency of single epitope variants in a cloned rabies virus seed was approximately 1:10,000. Animals were not or only partially protected when challenged with the parent virus or with another variant, but were fully protected against challenge with the virus used for immunization. Variants were also detected among seven street viruses obtained from fatal cases of human rabies. Animals immunized with standard rabies vaccine were protected against challenge with some but not all street rabies variants. A comparative antigenic analysis between vaccine strain and challenge virus by means of monoclonal antiglycoprotein antibodies showed a slightly closer degree of antigenic relatedness between vaccine and challenge strain in the combinations where vaccination resulted in protection. It remains unknown, however, whether these apparently minor antigenic differences in the glycoproteins account for the varying degrees of protection. The results of this study clearly indicate that the selection of vaccine strains and the methods used to evaluate the potency of rabies vaccines need to be revised.