Compounds inhibiting DNA repair and synthesis are expected to act synergistically with BCNU, a standard agent in the therapy of glioblastoma multiforme, and improve survival of patients with malignant gliomas. Ribonucleotide reductase (EC1.17.4.1; RR) catalyzes the rate-limiting step in DNA synthesis and plays a critical role in maintaining crucial substrates for DNA repair. We have studied the effects of Didox, an inhibitor of RR on 9L glioma cells in combination with BCNU. We analyzed intracellular dNTP pools and found that Didox significantly depleted the intracellular dNTP concentrations. Experiments using cytotoxicity, growth inhibition and clonogenic assays showed significant synergism of Didox and BCNU. Combination regimens using synchronous administration demonstrated highest cytotoxicity. We have also identified altered gene expression in a number of DNA repair related enzymes after BCNU treatment using large-scale cDNA arrays. The coadministration with Didox could reverse the expression of some of the overexpressed repair gene suggesting possible pathways to circumvent the developing resistance in 9L glioma cells against BCNU. These results introduce the combination of Didox and BCNU as a viable alternative for the treatment of malignant gliomas.