A cDNA copy of the RNA gene that encodes the nucleoprotein N of rabies virus Evelyn-Rokitnicki-Abelseth strain was cloned into baculovirus. The recombinant baculovirus expressed the N protein abundantly in Spodoptera frugiperda cells. The N protein was extracted from infected Spodoptera frugiperda cells and purified to near homogeneity by affinity chromatography. The purified N protein reacted with 31 of 32 monoclonal antibodies that recognize native rabies virus ribonucleoprotein. Like the ribonucleoprotein, the purified N protein was a major antigen capable of inducing virus-specific helper T cells. Priming of mice with the purified N protein prior to a booster inoculation with inactivated Evelyn-Rokitnicki-Abelseth virus vaccine resulted in a 20-fold increase in the production of virus-neutralizing antibodies. After immunization with the purified N protein, mice developed a strong anti-ribonucleoprotein antibody response and were protected against a lethal challenge of rabies virus. These data indicate that the N protein expressed in insect cells is antigenically and immunogenically comparable to the authentic rabies virus ribonucleoprotein and therefore represents a potential source of an effective and economical vaccine for large-scale immunization of humans and animals against rabies.