Gemcitabine (difluorodeoxycytidine; dFdCyd) is a potent radiosensitizer, noted for its ability to enhance cytotoxicity with radiation at noncytotoxic concentrations in vitro and subchemotherapeutic doses in patients. Radiosensitization in human tumor cells requires dFdCyd-mediated accumulation of cells in S phase with inhibition of ribonucleotide reductase, resulting in ≥80% deoxyadenosine triphosphate (dATP) depletion and errors of replication in DNA. Less is known of the role of specific DNA replication and repair pathways in the radiosensitization mechanism. Here the role of homologous recombination (HR) in relationship to the metabolic and cell cycle effects of dFdCyd was investigated using a matched pair of CHO cell lines that are either proficient (AA8 cells) or deficient (irs1SF cells) in HR based on expression of the HR protein XRCC3. The results demonstrated that the characteristics of radiosensitization in the rodent AA8 cells differed significantly from those in human tumor cells. In the AA8 cells, radiosensitization was achieved only under short (≤4 h) cytotoxic incubations, and S-phase accumulation did not appear to be required for radiosensitization. In contrast, human tumor cell lines were radiosensitized using noncytotoxic concentrations of dFdCyd and required early S-phase accumulation. Studies of the metabolic effects of dFdCyd demonstrated low dFdCyd concentrations did not deplete dATP by ≥80% in AA8 and irs1SF cells. However, at higher concentrations of dFdCyd, failure to radiosensitize the HR-deficient irs1SF cells could not be explained by a lack of dATP depletion or lack of S-phase accumulation. Thus, these parameters did not correspond to dFdCyd radiosensitization in the CHO cells. To evaluate directly the role of HR in radiosensitization, XRCC3 expression was suppressed in the AA8 cells with a lentiviral-delivered shRNA. Partial XRCC3 suppression significantly decreased radiosensitization [radiation enhancement ratio (RER) = 1.6 ± 0.15], compared to nontransduced (RER = 2.7 ± 0.27; P = 0.012), and a substantial decrease compared to nonspecific shRNA-transduced (RER = 2.5 ± 0.42; P = 0.056) AA8 cells. Although the results support a role for HR in radiosensitization with dFdCyd in CHO cells, the differences in the underlying metabolic and cell cycle characteristics suggest that dFdCyd radiosensitization in the nontumor-derived CHO cells is mechanistically distinct from that in human tumor cells.