It has previously been shown that the capacity to continue energy production by either oxidative or glycolytic metabolism is a pre-requisite for cellular survival at elevated temperatures. Since rhodamine 123 is a specific mitochondrial binding agent that inhibits oxidative phosphorylation, the present experiments were carried out to examine the influence of cell exposure to the drug which reduces the energy production at elevated temperatures. Exposure of HeLa cells to rhodamine 123 (10 micrograms/ml) up to 4 h was not cytotoxic in the presence or absence of glucose in the medium at 37 degrees C. Prolonged exposure of cells to the drug up to 24 h was quite cytotoxic when glucose was removed from the medium. When cells were exposed to rhodamine 123 at elevated temperatures (up to 42 degrees C) for 4 h, there was a pronounced enhancement of cell kill in the glucose-deprived cells. No enhanced effects of heat were seen in the glucose-fed cells. Hyperthermic treatment under the acidic pH did not further enhance the cytotoxic effects of heat in the glucose-deprived cells; instead, the cell kill under the influence of reduced pH was far less than the cytotoxicity observed at pH 7.4. Selective enhancement of hyperthermic cytotoxicity in glucose-deprived cells by rhodamine 123 appears to be in agreement with the concept that cell kill by heat is dependent on the critical level of cellular energy equilibrium.