The effect of hyperthermia on the functional activity of murine cytotoxic thymus-derived lymphocytes (CTL's) has been investigated. CTL's were generated in vitro in mixed leukocyte cultures (MLC's) and assayed for cytotoxicity on 51Cr-labeled allogeneic P-815 mastocytoma cells. Exposure of MLC cells to temperatures of 43 degrees C or above rapidly inactivated CTL function. The degree of inactivation depended on temperature and time of exposure to heat. CTL function was much more sensitive to hyperthermia than was the reproductive capacity of tumor target cells (assessed by a cloning assay), and the heat sensitivity of CTL's appeared to depend on their stage of differentiation. CTL function dramatically increased when MLC cells were incubated at 37 degrees C after exposure to hyperthermia or when a total exposure to heat was split into two equal fractions separated by an interval at 37 degrees C. These latter findings were operationally analogous to the repair of potentially lethal and sublethal radiation damage, respectively, and suggested that further studies are required to assess the complex interrelationships between hyperthermia, cell-mediated immunity, and tumor cell survival.