The radiosensitizing potential of gemcitabine (2',2'-difluoro-2'-deoxycytidine) was studied in combination with pulsed low dose-rate irradiation. The experiments were carried out with a human lung carcinoma cell line SW1573. These were irradiated at pulsed low dose rate (p-LDR); the average dose rate was 1 Gy/h. In the experiments with gemcitabine, this drug was applied for 24 h at a concentration of 10 nM prior to irradiation. The response of the cells to treatment was tested by using the standard clonogenic assay. Next to the cell-killing effects, damage to chromosomes was also assayed by using by whole chromosome Fluorescent In Situ Hybridization (FISH). Damage in chromosomes 2 and 18 was visualized by whole chromosome FISH and scored according to the PAINT method. A clear enhancement of the effects of radiation on cell survival was observed by preincubation of the cells with gemcitabine. The enhancement factor obtained from the p-LDR data was 1.7, which is much lower than the enhancement factor of 2.9 at high-dose rate. We did not observe a consistent increase in color junctions concomitant with radiosensitization. In chromosome 2, a small increase, and in chromosome 18, a decrease, in the number of color junctions was observed after radiation combined with gemcitabine compared to irradiation alone. These differences were not statistically significant. However, for the (unstable) acentric chromosome fragments from both chromosomes, significant changes were observed: In the case of chromosome 2, an increase, and in the case of chromosome 18, a decrease. So these results indicate that gemcitabine has no large and consistent effect on the repair of genomic lesions that induce secondary chromosome breaks. Although it is clear that gemcitabine-induced radiosensitization can be expected when it is combined with brachytherapy, as with radiation at a high-dose rate, the mechanism of radiosensitization is so far not evident, and further experiments will be needed to elucidate this.