Vaccine design against HIV-1 is complicated both by the latent aspects of lentiviral infection and the diversity of the virus. The type of vaccine approach used is therefore likely to be critically important. In general, vaccination strategies have relied on the use of live attenuated material or inactivated/subunit preparations as specific immunogens. Each of these methodologies has advantages and disadvantages in terms of the elicitation of broad cellular and humoral immune responses. Although most success has been achieved with live attenuated vaccines, there is a conceptual safety concern associated with the use of these vaccines for the prevention of human infections. In contrast, subunit or killed vaccine preparations enjoy advantages in preparation and conceptual safety; however, their ability to elicit broad immunity is more limited. In theory, inoculation of a plasmid DNA that supports in vivo expression of proteins, and therefore presentation of the processed protein antigen to the immune system, could be used to combine the features of a subunit vaccine and a live attenuated vaccine. We have designed a strategy for intramuscular DNA inoculation to elicit humoral and cellular immune responses against expressed HIV antigens. Uptake and expression are significantly enhanced if DNA is administered in conjunction with the facilitating agent bupivacaine-HCl. Using this technique we have demonstrated functional cellular and humoral immune responses against the majority of HIV-1 encoded antigens in both rodents and non-human primates.