DNA vaccination offers the advantages of viral gene expression within host cells without the risks of infectious virus. Like viral vaccines, DNA vaccines encoding internal influenza virus proteins can induce immunity to conserved epitopes and so may defend the host against a broad range of viral variants. CD8(+) cytotoxic T lymphocytes (CTL) have been described as essential effectors in protection by influenza nucleoprotein (NP), although a lesser role of CD4(+) cells has been reported. We immunized mice with plasmids encoding influenza virus NP and matrix (M). NP + M DNA allowed B6 mice to survive otherwise lethal challenge infection, but did not protect B6-beta(2)m(-/-) mice defective in CD8(+) CTL. However, this does not prove CTL are required, because beta(2)m(-/-) mice have multiple immune abnormalities. We used acute T cell depletion in vivo to identify effectors critical for defense against challenge infection. Since lung lymphocytes are relevant to virus clearance, surface phenotypes and cytolytic activity of lung lymphocytes were analyzed in depleted animals, along with lethal challenge studies. Depletion of either CD4(+) or CD8(+) T cells in NP + M DNA-immunized BALB/c mice during the challenge period did not significantly decrease survival, while simultaneous depletion of CD4(+) and CD8(+) cells or depletion of all CD90(+) cells completely abrogated survival. We conclude that T cell immunity induced by NP + M DNA vaccination is responsible for immune defense, but CD8(+) T cells are not essential in the active response to this vaccination. Either CD4(+) or CD8(+) T cells can promote survival and recovery in the absence of the other subset.