The lytic activity of splenocytes from C3H mice immunized with a highly attenuated line of Langat virus [tick-borne encephalitis (TBE) complex] was determined by the in vitro 51Cr release assay on TBE virus (western subtype)-infected target L929 cells. After the spleen cell suspensions were depleted of T cells with anti-mouse theta serum, or of B cells with anti-mouse immunoglobulin, the cytotoxic effect was found dependent on the presence of T lymphocytes. During immunization with Langat virus, cytotoxic T lymphocytes were generated, specific for both the foreign virus-specified antigen and the self component of the host system studied, coded by the K allele of the H-2 complex. The peak of T lymphocyte cytotoxic response was attained on the 6th day after administration of the live virus. T lymphocytes from mice, single-shot immunized with a flavivirus, displayed distinct cross-reactive lysis when studied on target cells infected with other flaviviruses but not on cells infected with an alphavirus.