A dengue (DEN) virus type 3 DNA vaccine expressing pre-membrane and envelope genes was tested for immunogenicity and protective efficacy in Aotus monkeys. Five of six vaccinated animals demonstrated moderate DEN-specific antibody responses as measured by ELISA and virus neutralization in vitro. By contrast, none of the six control animals developed detectable anti-DEN antibodies. When five vaccinated animals were challenged with live DEN-3 virus and viremia determined by PCR amplification of viral RNA in serum samples, one animal was completely protected and two were partially protected as indicated by a decrease in mean days of viremia. The results demonstrate the ability of the DEN-3 DNA vaccine to elicit a neutralizing antibody response and to partially protect against live virus challenge. These findings support the inclusion of this construct in a tetravalent DNA vaccine.