During selection for methotrexate resistance, SV40-transformed human skin fibroblasts from patients with ataxia telangiectasia (A-T) underwent amplification of the dihydrofolate reductase (DHFR) gene, experienced nearly complete loss of the integrated SV40 sequences and showed a 3.6-fold increase in Ki-ras gene copy number. Over a period of months methotrexate-resistant (MTXr) A-T subclones were obtained, which were able to grow in progressively increasing MTX concentrations up to 100 microM. The ED50 values determined as the effective dose of MTX causing 50% growth inhibition in comparison to control cells increased from 3 x 10(-2) microM for MTXs AT5BI-VA cells to 250 microM MTX for the MTXr AX100 subclone. In contrast, human skin fibroblasts of healthy individuals did not show DHFR gene amplification and loss of SV40 sequences under comparable conditions and were unable to grow in MTX concentrations greater than 1 microM. Gene amplification and loss of DNA sequences are features underlying the genomic instability known to be a characteristic property of A-T cells and being probably responsible for the high cancer incidence in these patients.