Although the ability of serum-neutralizing antibodies to protect against picornavirus infection is well established, the contribution of cell-mediated immunity to protection is uncertain. Using major histocompatibility complex class II-deficient (RHA^/) mice, which are unable to mediate CD4⁺ T-lymphocyte-dependent humoral responses, we demonstrated antibody-independent protection against lethal encephalomyocarditis virus (EMCV) infection in the natural host. The majority of RHA^/ mice inoculated with 10⁴ PFU of attenuated Mengo virus (vMC₂₄) resolved infection and were resistant to lethal challenge with the highly virulent, serotypically identical cardiovirus, EMCV. Protection in these mice was in the absence of detectable serum-neutralizing antibodies. Depletion of CD8⁺ T lymphocytes prior to lethal EMCV challenge ablated protection in vMC₂₄-immunized RHA^/ mice. The CD8⁺ T-lymphocyte-dependent protection observed in vivo may, in part, be the result of cytotoxic T-lymphocyte (CTL) activity, as CD8⁺ T splenocytes exhibited in vitro cytolysis of EMCV-infected targets. The existence of virus-specific CD8⁺ T-lymphocyte memory in these mice was demonstrated by increased expression of cell surface activation markers CD25, CD69, CD71, and CTLA-4 following antigen-specific reactivation in vitro. Although recall response in vMC₂₄-immunized RHA^/ mice was intact and effectual shortly after immunization, protection abated over time, as only 3 of 10 vMC₂₄-immunized RHA^/ mice survived when rechallenged 90 days later. The present study demonstrating CD8⁺ T-lymphocyte-dependent protection in the absence of serum-neutralizing antibodies, coupled with our previous results indicating that vMC₂₄-specific CD4⁺ T lymphocytes confer protection against lethal EMCV in the absence of prophylactic antibodies, suggests the existence of nonhumoral protective mechanisms against picornavirus infections.