Lonidamine is a potent inhibitor of spermatogenesis and a hyperthermic sensitizer. The previous study of lonidamine and radiation using two murine tumors demonstrated that tumor cure rates were significantly increased by radiation and concomitant lonidamine. In an effort to determine the radiobiologic factors involved with the potentiating effect of radiation by lonidamine, a series of cell culture studies were carried out using multicellular tumor spheroids (MTS) of HeLa Cells. When the MTS were treated with lonidamine in combination with fractionated irradiation, remarkable enhancement of growth inhibition was observed at the drug concentration of 10 micrograms/ml. On the other hand, there was no demonstrable enhancement of growth inhibition induced by a single dose of irradiation. Although the present findings would be consistent with the inhibitory action of potentially lethal damage repair of radiation by the drug, an alternative possibility is that the cells that have received the combined treatment have undergone a metabolic change, which has altered their sensitivity to the growth inhibitory effects of lonidamine. Based on the studies reported here and in mice, it is suggested that continued drug exposure over a prolonged period may provide an enhanced therapeutic effect, even in tumor varieties where the drug has no apparent antitumor activity on nonirradiated cells.