Imexon is an iminopyrrolidone aziridine which previously has shown activity against a variety of human cancer types, including multiple myeloma and pancreatic adenocarcinoma. Recently, mechanistic studies in the MIA PaCa-2 human pancreatic cancer cell line have demonstrated binding to sulfhydryls, build-up of reactive oxygen species (ROS), perturbations in mitochondrial membrane potential (MMP), and activation of caspases 3, 8 and 9. Because imexon binds sulfhydryls and generates ROS, it was hypothesized that imexon would have considerable activity against pancreatic cancer by promoting oxidative stress in cells which are already oxidatively challenged and in combination with gemcitabine by interacting with key sulfhydryl-dependent enzymes involved with gemcitabine metabolism. In vitro anti-tumor activity of imexon and gemcitabine was evaluated in PANC-1, MIA PaCa-2, MutJ, and BxPC-3 human pancreatic cancer cell lines. Interactions between imexon and gemcitabine were assessed with simultaneous drug exposure at a fixed (imexon: gemcitabine) ratio using median effect analysis. The PANC-1, MutJ, and BxPC-3 cells demonstrated synergy with combination treatment. Severe combined immune deficient (SCID) mice bearing PANC-1 cells treated with imexon and gemcitabine demonstrated tumor growth inhibition and regression. Imexon inhibited ribonucleotide reductase (RNR) at drug concentrations ≥100 μMol. This is similar to the selective RNR inhibitor hydroxyurea, suggesting that imexon may enhance gemcitabine-mediated inhibition of RNR as a mechanism of synergy. An S phase accumulation of PANC-1 cells occurred at ≥300 μMol imexon at 24 hr. This was associated with a ≥2-fold increase of radiolabeled gemcitabine incorporation into PANC-1 DNA at ≥100 μMol imexon. Therefore the mechanisms of synergy between imexon and gemcitabine appear to include: (1) cell cycle arrest in S-phase, and (2) inhibition of RNR. Both actions would increase the uptake of the active metabolite, gemcitabine-triphosphate, (GEM-TP), into DNA. Arresting cells in S-phase would increase the time of cellular incorporation of deoxynucleotides, including GEM-TP, into DNA. Similarly, RNR inhibition reduces the availability of normal deoxynucleotides which compete with GEM-TP for incorporation. Overall, these data demonstrate that imexon is uniquely synergistic with gemcitabine in vitro and in vivo and support the rationale for combining the agents in clinical trials for the treatment of pancreatic cancer.