DNA vaccines have the potential of giving rise to a potent cell-mediated immune response by inducing intracellular synthesis and subsequent antigenic presentation of encoded antigens. We have tested a DNA vaccine specific for human immunodeficiency virus type 1 (HIV-1) by the injection of animals with expression plasmids encoding the HIV-1 envelope protein and the Rev regulatory protein. Injection of both plasmids into mice, rabbits, or macaques was found to induce high levels of specific antibodies capable of efficiently inhibiting both HIV-1 infection and envelope-mediated cell fusion. A readily detectable delayed-type hypersensitivity (DTH) response was demonstrable in injected mice and lymphocytes derived from these proliferated in response to an HIV-1 envelope V3 loop-specific peptide. Interestingly, the injected mice or macaques also developed a strong cytotoxic T lymphocyte (CTL) response against target cells pulsed with the V3 peptide. Taken together, these data demonstrate that injection of HIV-1 gene expression plasmids can induce potent humoral and cell-mediated immune responses and suggest that DNA vaccines may prove to be significantly beneficial as a means of immunizing against HIV-1.