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Cytotoxic CD8(+) T cells control acute viremia in many viral infections. However, most viruses that establish chronic infections evade destruction by CD8(+) T cells, and regulatory T cells (Treg) are thought to be involved in this immune evasion. We have infected transgenic mice, in which Treg can be selectively depleted, with Friend retrovirus (FV) to investigate the influence of Treg on pathogen-specific CD8(+) T-cell responses in vivo. We observed that Treg expansion during acute infection was locally defined to organs with high viral loads and massive activation of virus-specific effector CD8(+) T cells. Experimental ablation of Treg resulted in a significant increase of peak cytotoxic CD8(+) T-cell responses against FV. In addition, it prevented the development of functional exhaustion of CD8(+) T cells and significantly reduced FV loads in lymphatic organs. Surprisingly, despite the massive virus-specific CD8(+) T-cell response after temporary Treg depletion, no evidence of immunopathology was found. These results demonstrate the important role of Treg in controlling acute retrovirus-specific CD8(+) T-cell responses, and suggest that temporary manipulation of Treg might be a possible therapeutic approach in chronic infectious diseases.