Although 5-fluorouracil (5-FU) is commonly used in conjunction with radiotherapy to treat gastrointestinal malignancies, the molecular mechanisms underlying the clinically observed therapeutic advantage of this combination of agents have not been clearly established. The present in vitro studies addressed the possibility that the radiosensitization of log-phase cultured human colon adenocarcinoma cells by postirradiation administration of 5-FU was accompanied by an interference either with the rejoining of radiation-induced DNA double-strand breaks (DSB's) or with recovery from potentially lethal damage (PLD). Significantly more killing was observed in cells exposed to gamma-rays (1-6 Gy) and then treated with 5-FU (100 micrograms/mL; 0.77 mM) for 1 hr at 37 degrees C than in cells given gamma-rays but not 5-FU; essentially, the survival curve shoulder was removed. DSB rejoining measured using the neutral filter elution method after exposure to 25 Gy was identical regardless of whether 5-FU (100 micrograms/mL) was present during the repair period; thus, radiosensitization by this high-concentration postirradiation 5-FU protocol does not appear to be a result of interference with the overall rate of ligation of gamma-ray-induced DSB's. The effect of 5-FU on the ability of log-phase cells to recover from that sector of PLD that can be expressed by postirradiation incubation with hypertonic (0.5 M) salt solution (HSS) was also examined. When irradiated cells were treated with 5-FU during their recovery period and then incubated with HSS, no clonogenic cells survived. Therefore, although it was not possible to assess the actual kinetics of recovery from gamma-ray-induced PLD in 5-FU-treated cells, the drug clearly altered the metabolism or structure of the cells such that their susceptibility to HSS was markedly enhanced.