Immunization with more than one immunogen (co-immunization) is an efficient regimen to induce immunity to multiple antigens. However, immune interference has been reported using multi-plasmid DNA immunizations. HIV-1 envelope (Env) and Gag gene products are the predominant immunogens used in current AIDS vaccines, although, few studies have evaluated possible immune interference when these two antigens are co-administered. Therefore, in this study, immune interference during co-inoculation was examined using DNA vaccines expressing lentiviral Envs and Gag from gene sequences optimized for efficient expression in mammalian cells (codon-optimized). BALB/c mice vaccinated in separate hind legs with each plasmid individually elicited high titer immune responses, however, when HIV-1 Envgp120 and HIV-1 Gagp55 DNA plasmids were co-inoculated, there was a reduction in the immune responses elicited to HIV-1 Gagp55. To determine if the anti-HIV-1 Gagp55 immune interference was specific to HIV-1 Envgp120, mice were coimmunized with plasmids expressing the surface envelope protein from two additional lentiviruses, Envgp130-SIV or Envgp90-EIAV, or a soluble form of hemagglutinin (sHA) from influenza virus and HIV-1 Gagp55- or SIV Gagp55-DNA. Interestingly, there was no reduction in anti-HIV-1 Gagp55 immune responses using other lentiviral envelopes or the influenza sHA. Also, none of the lentiviral envelopes reduced anti-SIV Gagp55 immune responses during co-immunization. Therefore, anti-HIV-1 Gag immune interference appears specific to co-immunizations with HIV-1 Envgp120 and may involve a yet undefined immunological mechanism(s).