Cationic dyes such as EDKC, which preferentially accumulate in malignant cell mitochondria, permit selective photodamage and dark toxicity to these organelles. The resultant inhibition of oxidative phosphorylation and decrease in intracellular ATP should strongly potentiated thermal injury, even at temperatures which are relatively innocuous by themselves. In human squamous carcinoma (FaDu), murine melanoma (B-16) or non-malignant monkey kidney (CV-1) cells, we examined the effect of combining treatment with EDKC ± light together with mild, 42°C hyperthermia. Heat alone for up to 60 min had no effect on any of the cell lines, and EDKC + light + heat was not toxic to the non-malignant CV-1 cells. Hyperthermia synergistically enhanced photodamage in malignant cells, with survival decreasing more than 200-fold after 20 min of heat to B-16 cells or 60 min to FaDu cells. The heating also increased dark toxicity of the dye, decreasing survival of FaDu cells by more than 10-fold after treatment with 10-7 M EDKC and 60 minutes at 42°C. The sequence of application of light and heat was important, with much greater synergy for heat after irradiation.