Conventional CD4(+) T cells play an important role in viral immunity. In most virus infections they provide essential help for anti-viral B and T cell responses. In chronic infections, including HIV, an expansion of regulatory T cells (Tregs) has been demonstrated, which can suppress virus-specific CD4(+) T cell responses in vitro. However, the suppressive activity of Tregs on effector CD4(+) T cells in retroviral infection is less well documented in vivo. We took advantage of a transgenic mouse in which Tregs can be selectively depleted to determine the influence of such cells on retrovirus-specific CD4(+) T cell responses during an ongoing infection. Mice were infected with Friend retrovirus (FV) and Tregs were depleted during the acute phase of the infection. In non-depleted mice activated CD4(+) T cells produced Th1-type cytokines but did not exhibit any anti-viral cytotoxicity as determined in an MHC class II restricted in vivo CTL assay. Depletion of Tregs significantly increased the numbers of virus-specific CD4(+) T cells and improved their cytokine production, whereas it induced only very little CD4(+) T cell cytotoxicity. However, after dual depletion of Tregs and CD8(+) T cells conventional CD4(+) T cells developed significant cytotoxic activity against FV epitope-labeled target cells in vivo and contributed to the control of virus replication. Thus, both Tregs and CD8(+) T cells influence the cytotoxic activity of conventional CD4(+) T cells during an acute retroviral infection