The importance of lymphotoxin alpha (LTalpha) in lymphoid organogenesis is well established. Although LTalpha has been implicated in the pathogenesis of T-cell-mediated immunopathologies, the requirement for LTalpha in T-cell activation and effector function in vivo is not well understood. To determine the role of LTalpha in T-cell activation in vivo, we compared the generation of antigen-specific T-cell responses between wild type (+/+) and LTalpha-deficient (LTalpha(-/-)) mice during an acute infection with lymphocytic choriomeningitis virus (LCMV). Our studies showed that LCMV-infected LTalpha(-/-) mice had a profound impairment in the activation and expansion of virus-specific CD8 T cells in the spleen, as determined by cytotoxicity assays, intracellular staining for gamma interferon, and staining with major histocompatibility complex class I tetramers. Further, the nonlymphoid organs of LTalpha(-/-) mice also contained substantially lower number of LCMV-specific CD8 T cells than those of +/+ mice. Greatly reduced virus-specific CD8 T-cell responses in LTalpha(-/-) mice led to a defect in LCMV clearance from the tissues. In comparison to that in +/+ mice, the activation of LCMV-specific CD4 T cells was also significantly attenuated in LTalpha(-/-) mice. Adoptive transfer experiments were conducted to determine if abnormal lymphoid architecture in LTalpha(-/-) mice caused the impairment in the activation of LCMV-specific T-cell responses. Upon adoptive transfer into +/+ mice, the activation and expansion of LCMV-specific LTalpha(-/-) T cells were restored to levels comparable to those of +/+ T cells. In a reciprocal cell transfer experiment, activation of +/+ T cells was significantly reduced upon transfer into LTalpha(-/-) mice. These results showed that impairment in the activation of LCMV-specific T cells in LTalpha(-/-) mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LTalpha(-/-) T cells.