Exposure of L929 cells to hyperthermia in the presence of 2-cyanocinnamic acid, an inhibitor of mitochondrial pyruvate transport, markedly enhanced killing at temperatures as low as 41 degrees C. The inhibitor also reduced the oxidation of both glucose and exogenous pyruvate while increasing lactate production from glucose but not from pyruvate. These results are consistent with previous observations that glucose and pyruvate enhance survival after hyperthermia and point to a role for pyruvate in protecting against hyperthermic cytotoxicity. Heat shock protein synthesis was also inhibited by 2-cyanocinnamic acid under some conditions, suggesting that a function of pyruvate may also be involved in heat shock protein induction.