Rabies prevention and appropriate population management of free-ranging animals have an important role to play in the eventual elimination of rabies in dogs. An effective sterilant based on rabies vaccines has the potential to create a supportive measure of public acceptability and to reduce associated clinic visit costs. We inserted the coding sequence of gonadotropin-releasing hormone (GnRH) into different locations within the rabies virus ERA glycoprotein (G) gene, and demonstrated that the amino terminus (N), antigenic site IIa, and the junction between the ecto- and cytoplasmic domains (C) of the G were suitable sites for GnRH insertion. The rescued recombinant rabies viruses ERA-N-GnRH and ERA-C-GnRH grew as well as the parental ERA virus, reaching 1x10(9)ffu/ml in cell culture. Insertion and expression of the GnRH were stable in the viruses after multiple passages in vitro. To increase immunogenicity of the GnRH peptide, two copies of GnRH, aligned in tandem, were fused to the N terminus of the G. The recombinant rabies virus ERA-N-2GnRH was recovered and grown to high titers in cell culture. All GnRH-carrying rabies viruses induced antibodies against GnRH in immunized mice and protected 100% of the animals after rabies virus challenge. The recombinant viruses reacted strongly with the serum from a GonaCon-immunized animal. The GnRH-carrying rabies viruses have significant potential in rabies and animal population control.