Hyperthermia is being tested as an adjuvant to chemotherapy for clinical use. We elected to study the interaction of heat at 43 degrees C with actinomycin D (AMD) (0.5 microgram/ml) in tissue culture using plateau phase Chinese hamster cells. The simultaneous administration of 43 degrees C and AMD produces more than additive cytotoxicity if the duration of exposure is less than 30 minutes; however, this is quickly reversed with longer exposures with the cells developing resistance to further cytotoxicity of AMD. If heat (43 degrees C) is applied before AMD exposure, the cells also are rendered insensitive, with a greater protection observed for longer periods of heating. For example, for cells heated for 2 hours at 43 degrees C and then exposed to AMD (0.5 microgram/ml for 2 hours), cytotoxicity to AMD is decreased by a factor of 10. Heat-induced resistance to AMD persists for at least 18 hours before full recovery of AMD effect returns. The application of heat following AMD exposure also protects against the cytotoxicity of AMD. Studies using 3H-AMD demonstrate that the resistance does not correlate with reduced membrane permeability to AMD of heated cells. Attention must be given to the timing of hyperthermia when used clinically as adjuvant therapy in patients receiving AMD.