A nucleic acid vaccine for dengue-2 virus was developed, consisting of a plasmid DNA vector with the pre-membrane (prM) and envelope (E) genes expressed from a cytomegalovirus promoter. The DNA was adsorbed onto gold microspheres for administration by a gene gun. Expression was demonstrated by transfection of mouse cells in culture where the prM and E antigens were detected intracellularly, and the E antigen was detected in the culture supernatant fluid, similar to a natural infection. The vaccine elicited neutralizing antibodies to dengue-2 virus and antigen-specific cytotoxic T lymphocyte responses in mice. Several vaccination regimens were evaluated in rhesus macaques for the ability to elicit neutralizing antibodies and protect against viremia after challenge with live dengue-2 virus. Neutralizing antibodies were measured in three of three animals that received four 2-microg doses of DNA and in two of six animals that received two 1-microg doses. No antibodies were detected in three animals that received a single 1-microg dose. When dengue virus challenge was performed one month after vaccination, the three animals that received four 2-microg doses exhibited 0, 0, and 1 day of viremia compared with unimmunized controls which exhibited 4, 4, and 6 days of viremia. Three animals that received two 1-microg doses also exhibited 0, 0, and 1 day of viremia, whereas three animals that received a single 1-microg dose exhibited 2, 3, and 5 days of viremia compared with unimmunized controls, which exhibited 4 days of viremia each. When challenge was performed 7 months after vaccination, three animals that received two 1-microg doses exhibited 0, 3, and 5 days of viremia compared with unimmunized controls, which exhibited 4, 5, and 9 days of viremia. These results suggest that a regimen consisting of two 1-microg doses of DNA can confer satisfactory protection at one month, but not at seven months, after vaccination. Long-term protection following DNA vaccination may require revaccination, higher doses of DNA, or a vaccine that contains additional epitopes or adjuvants.