During infection with Banzi virus, splenic thymus-derived cells of C3H/RV mice developed immunologically specific cytotoxicity for Banzi virus-infected, C3H-derived L 929 fibroblasts. Cytotoxic activity was detected by six days after viral inoculation, was maximal by eight days, and decreased to background levels by 16 days. There was no consistently significant difference in Banzi virus-specific cytotoxicity of spleen cells from C3H/RV and C3H/He mice, which are genetically resistant and susceptible, respectively, to the lethal flaviviral encephalitis. Therefore, the ability of the host to develop killer cells did not, in itself, lead to protection from lethal infection. These experiments are, to our knowledge, the first demonstration of cell-mediated cytotoxicity to a flavivirus.