Interleukin-18 (IL-18) is a proinflammatory cytokine that promotes natural killer (NK) and T-cell activation. Several poxviruses, including vaccinia virus (VV), encode a soluble IL-18-binding protein (IL-18bp). The role of the VV IL-18bp (gene C12L) in vivo was studied with wild-type (vC12L), deletion mutant (vDeltaC12L), and revertant (vC12L-rev) viruses in a murine intranasal model of infection. The data show that vDeltaC12L was markedly attenuated, characterized by a mild weight loss and reduced virus titers in lungs, brain, and spleen. Three days after infection, NK cytotoxic activity was augmented in the lung, spleen, and mediastinal lymph nodes (MLNs) of vDeltaC12L-infected mice compared to controls. Seven days after infection, vDeltaC12L-infected mice displayed heightened VV-specific cytotoxic T-lymphocyte (CTL) responses in the lungs, spleen, and MLNs. Gamma interferon (IFN-gamma) levels were also dramatically elevated in lavage fluids and cells from lungs of mice infected with vDeltaC12L. Finally, we demonstrate that IL-18 is produced in vitro and in vivo after VV infection. Taken together, these data demonstrate a role for the vIL-18bp in counteracting IL-18 in both the innate and the specific immune response to VV infection and indicate that the ability of IL-18 to promote vigorous T-cell responses (cytotoxic activity and IFN-gamma production) is a critical factor in the accelerated clearance of the vDeltaC12L mutant.