Direct DNA inoculation induces immune responses through the delivery of nonreplicating transcription units that drive the synthesis of specific foreign proteins within the inoculated host. These proteins are processed within host cells and through association with relevant MHC antigens that can become the subject of immune surveillance and elicit immune responses against pathogens. Direct introduction of DNA into mice has been reported to be antigenic as demonstrated by the use of this technique to develop immune responses against human growth hormone, influenza proteins, as well as HIV-1 proteins. Most recently the demonstration of the use of this technology to produce anti-HIV-1 immune responses has been reported in nonhuman primates. Accordingly a more detailed analysis of this technology could generate important insight into the generality of this approach for immune therapy or vaccine design. In this article we further our investigation of direct DNA inoculation as a tool for induction of relevant immune responses against HIV-1 in vivo. We demonstrate expression of HIV-1 antigens in the inoculated muscle of animals. Inoculated animals demonstrate significant cytotoxic T cell responses against HIV-1 antigen-expressing targets. Furthermore, using a novel challenge system, we demonstrate that the majority of immunized animals can reject lethal, HIV-1 antigen-expressing cell challenge in an antigen-specific manner. This technology has relevance for the development of immunization strategies against HIV as it provides for specific antigen production in vivo without the use of infectious agents.