Resistance to cytotoxic nucleoside analogues is a major problem in cancer treatment. The cellular mechanisms involved in this phenomenon have been studied for several years, and some factors have been identified. However, this resistance seems to be multifactorial and more studies are needed to gain better insight into this domain. For this purpose, we developed a gemcitabine-resistant cell line (MCF7 1K) from the human mammary adenocarcinoma MCF7 strain by prolonged exposure to gemcitabine in vitro. MCF7 1K cells are highly resistant to gemcitabine (533-fold) and cross-resistance is observed with araC (47-fold), triapine (14-fold), and hydroxyurea (6.7-fold). Quantitative real-time reverse transcription-PCR and Western blot analysis showed an increase in the gene and protein expression of the large subunit of ribonucleotide reductase, R1. Ribonucleotide reductase activity was also significantly increased in the gemcitabine-resistant cells. Study of genomic DNA showed 12-fold increase in R1 gene dosage in MCF7 1K cells. In contrast, the gene and protein expression of the small subunit of ribonucleotide reductase, R2, were not modified in this cell line. These results show that gemcitabine resistance can be associated with genetic modifications of target genes in malignant cells, and suggest that the large subunit of human ribonucleotide reductase is involved in the cellular response to gemcitabine.