alpha-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), was used to select two very highly drug-resistant cell lines, designated K562-DFMOr and V79-DFMOr. Both DFMO-resistant cell lines exhibited elevated ODC expression due to gene amplification. Moreover, the K562-DFMOr cells, but not the V79-DFMOr cells, had an elevated level of ribonucleotide reductase subunit R2 (R2) mRNA and an increased R2 gene copy number. By analysis of their electron paramagnetic resonance spectra, an increased level of the R2 protein was observed in the K562-DFMOr cells as compared to the wild type K562 cells. This is the first description of a DFMO-induced mutant cell line exhibiting coamplification of the genes for ODC and R2, and overexpression of their products. There was no coamplification of the N-myc protooncogene, which is located close to the ODC and R2 genes on human chromosome 2. The alterations exhibited by the K562-DFMOr cell line were shown to be stable for many passages and to convey resistance not only to DFMO but also to hydroxyurea, an inhibitor of ribonucleotide reductase and thus DNA replication. In the absence of the selective pressure exerted by DFMO, the V79-DFMOr cell line produced revertants by loss of ODC gene amplification within three passages. Coamplification of linked genes may turn out to be an important mechanism in the development of cross-resistance and should be considered when designing therapeutic strategies.