DNA immunization is being considered to augment, or even to supplant, more traditional methods of antiviral immunization. Different routes of administration lead to markedly different levels of marker protein expression, but only limited data are available concerning the antiviral responses induced by DNA inoculated by different routes, and their protective efficacy. In this report we evaluate antiviral immunity induced by inoculation of DNA by the intramuscular (i.m.) and intradermal (i.d.) routes, and make three novel observations. First, i.d. immunization is dose-dependent and, although not uniformly successful, can induce very high levels of cytotoxic T lymphocyte (CTL) activity, varying dependent on the vehicle in which the DNA is administered. Second, while antiviral immunity induced by i.m. DNA injection has been demonstrated by many groups, we show herein a marked difference in immunity depending on the muscle injected. Immunity induced by DNA injection of the anterior tibial muscle significantly exceeds that induced following injection of the quadriceps muscle as judged by three criteria, namely CTL induction, decrease in virus titer following nonlethal challenge, and survival following a normally lethal challenge dose of virus. Thirdly, we evaluate the local immune response induced following immunization with DNA encoding a viral antigen. We show that, when recipients are already immune to the encoded protein, a severe but localized inflammatory response may result.