L5 mouse fibroblast cells, cultured in various concentrations of 5-bromo-2′-deoxyuridine (BrdUrd) and/or methotrexate (MTX) for 48 hr, were exposed to X-rays, and the surviving fractions of the cells were estimated by their colony-forming ability. The radiosensitizing effect of BrdUrd was found to be dependent on the concentration of BrdUrd only within the range of 0.001 to 10 μg/ml. In the medium to which MTX, 0.005 μg/ml, was added, a synergistic increase in the radiosensitizing effect of BrdUrd was observed within the range of 0.01 to 10 μg/ml. Outside this range of BrdUrd concentrations, only an additive increase in radiosensitizing effect was seen. The optimum range of BrdUrd concentration for synergistic effect was 0.1 to 0.5 μg/ml. Radiosensitization of BrdUrd supplemented with MTX at varying concentrations was studied, and the optimum range of MTX concentration bringing about the maximum radiosensitizing effect of BrdUrd was found to be about 0.005 to 0.01 μg/ml. In conclusion, the enhancement of the radiosensitizing effect of BrdUrd was found to be dependent not only on the BrdUrd concentration but also on the MTX concentration. In this in vitro study with L5 cell culture, the optimum range of concentration of BrdUrd and MTX for maximum enhancement was 0.1 to 0.5 μg/ml and 0.005 to 0.01 μg/ml, respectively.