Bovine leukemia virus (BLV) is a complex B-lymphotrophic retrovirus of cattle and the causative agent of enzootic bovine leukosis. Serum antibody in infected animals does not correlate with protection from disease, yet only some animals develop severe disease. While a cytotoxic T-lymphocyte response may be responsible for directing BLV pathogenesis, this possibility has been left largely unexplored, in part since the lack of readily established cytotoxic target cells in cattle has hampered such studies. Using long-term naturally infected alymphocytic (AL) cattle, we have established the existence of cytotoxic T-lymphocyte response against BLV envelope proteins (Env; gp51/gp30). In vitro-expanded peripheral blood mononuclear (PBM) cell effector populations consisted mainly of ⁺ (>40%), CD4⁺ (>35%), and CD8⁺ (>10%) T lymphocytes. Specific lysis of autologous fibroblasts infected with recombinant vaccinia virus (rVV) delivering the BLV env gene ranged from 30 to 65%. Depletion studies indicated that ⁺ and not CD8⁺ T cells were responsible for the cytotoxicity against autologous rVVenv-expressing fibroblasts. Additionally, cultured effector cells lysed rVVenv-expressing autologous fibroblasts and rVVenv-expressing xenogeneic targets similarly, suggesting a lack of genetic restricted killing. Restimulation of effector populations increased the proportion of ⁺ T cells and concomitantly Env-specific cytolysis. Interestingly, culture of cells from BLV-negative or persistently lymphocytic cattle failed to elicit such cytotoxic responses or increase in ⁺ T-cell numbers. These results imply that cytotoxic ⁺ T lymphocytes from only AL cattle recognize BLV Env without a requirement for classical major histocompatibility complex interactions. It is known that ⁺ T lymphocytes are diverse and numerous in cattle, and here we show that they may serve a surveillance role during natural BLV infection.