CD4⁺ T cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4⁺ T cells have been described, whether those generated during the course of a viral infection play a role in virus control remains unknown. Here we show that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4⁺ T cells in the draining lymph node and liver of resistant mice have a cytotoxic phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4⁺ T cells have impaired virus control. Thus, perforin-dependent CD4⁺ T-cell killing of infected cells is an important mechanism of antiviral defense.