DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.