Acceleration of clonogen repopulation during fractionated irradiation after about 3 weeks has been demonstrated previously in FaDu human squamous cell carcinoma in nude mice (Petersen et al., Int. J. Radiat. Oncol. Biol. Phys. 51, 483-493, 2001). Selection of genetically distinct, rapidly proliferating clones might contribute to this phenomenon. To address this question, three sublines (R1-R3) were established from FaDu tumors that recurred locally after fractionated irradiation. The tumors were retransplanted and irradiated under clamp hypoxia with single doses or with 18 x 3 Gy within 18 days or 36 days, followed by graded top-up doses. The results were compared with data obtained after the same treatment schedules in the parental tumor line. Histologies, tumor volume doubling times, and potential doubling times of FaDu sublines R1-R3 were not different from those of the parental line. The radiation dose required to control 50% of the tumors (TCD(50)) after single-dose irradiation of 37-38 Gy was the same for the FaDu sublines R1-R3 and the parental tumor. The top-up TCD(50) values for the FaDu sublines R1-R3 after 18 fractions within 36 days were 14-17 Gy higher than those after 18 fractions within 18 days, indicating significant repopulation. The magnitude of this effect was not significantly different between the sublines R1-R3 or between these sublines and the parental FaDu tumors. The results indicate that selection of genetically distinct, rapidly proliferating clones does not contribute to the acceleration of repopulation during fractionated irradiation in poorly differentiated FaDu tumors.