A plasmid vector expressing the full-length rabies virus glycoprotein (G protein) under the control of the simian virus 40 (SV40) promoter has previously been shown to induce upon intramuscular (i.m.) inoculation into mice a specific B- and T-cell-mediated immune response and protection against challenge with a virulent strain of the virus. Here we tested two parameters that might affect the efficacy of this DNA vaccine. First, we replaced the SV40 promoter of the original vector with the early promoter derived from cytomegalovirus leaving all other parameters of the plasmid intact. Although upon transfection in vitro the two vectors showed a striking difference in their ability to cause stable expression of the rabies virus G protein, upon i.m. inoculation into mice both constructs induced comparable immune responses. Second, we constructed a vector that induces expression of a secreted form of rabies G protein by inserting a stop codon just upstream of the transmembrane domain of the rabies G protein gene. The immune responses to the DNA vaccines expressing the two different forms of the G protein, secreted and membrane bound, were compared and found to be similar in magnitude. The long-term effect of DNA vaccination was also investigated especially with regard to adverse immunological reactions such as the induction of unresponsiveness against rabies virus and the development of antibodies to DNA. DNA vaccination was found to induce long-lasting immunity to rabies virus without apparent negative side effects such as development of T cell tolerance or generation of anti-DNA antibodies.