Evidence is presented for the interaction of X irradiation, slightly toxic levels of chloroquine, and mild hyperthermia in the inactivation of colony-forming ability in asynchronous HeLa cells. A three-way interaction was observed which resulted in the potentiation of radiation-induced lethality. There was little evidence of toxicity in unirradiated cells incubated for 3 h with 0.1 mM chloroquine at either 37 or 41 degrees C. The radiopotentiation factor, which is similar to the dose modification factor, was determined from dose-response curves by relating the reciprocal of the slope (D0) of the reference survival curve to that of the survival curve of cells receiving the combined postirradiation treatment with chloroquine and mild hyperthermia. Radiopotentiation factors larger than 1.7 were obtained irrespective of whether the reference D0's were obtained from survival curves for cells irradiated at 37 degrees C without drug or from cells receiving postirradiation treatment with heat or drug only.