Lysosomes of cultured mammalian cells, derived from a transplantable murine mammary adenocarcinoma, irreversibly lose their capacity to accumulate the fluorescent dye acridine orange after hyperthermia. As acridine orange may be regaraded as a fluorescent probe of the internal pH of the lysosomes, we may conclude that the ability of lysosomes to maintain a low internal pH is destroyed by hyperthermia. The effects of hyperthermia on lysosome fluorescence and on cell survival show several similarities: in both cases hyperthermia is more effective at low pH, below pH 7.0, and CCP (carbonylcyanide-m-chlorophenylhydrazone) enhances effects at low pH, but has no clear effect at pH 8.0. This leads to the conclusion that effects on lysosomes are an important and early event in cellular injury caused by hyperthermia. The activation energy, however, obtained for the effects of hyperthermia on lysosome fluorecence is about a factor of two lower athan the activation energy reported for cell survival after hyperthermia. This suggests that the effect on lysosomes is not directly caused by hyperthermia but is triggered by some other hyperthermia-induced cellular damage.