A methotrexate-resistant subline, CCRF-CEM/R1, was selected stepwise from the human leukemic lymphoblast T-cell line, CCRF-CEM, and maintained in 0.2 microM methotrexate. The development of resistance to methotrexate (75-fold) was associated with a 20-fold increase of dihydrofolate reductase activity. The affinity of dihydrofolate reductase from the resistant cells for methotrexate did not vary significantly as compared to the enzyme from the parent cells. Southern blot analysis of DNA from parent and CCRF-CEM/R1 cells demonstrated amplification of the dihydrofolate reductase gene in the resistant line. Quantitative dot-blot DNA hybridization demonstrated the presence of about 18 reductase gene copies in the R1 cells. The human dihydrofolate reductase gene contained at least 4 intervening sequences and was about 30 kilobases in size. Northern blot analysis demonstrated an increase in dihydrofolate reductase messenger RNA species, the predominant message was 3.8 kilobases. Cytogenetic analysis of CCRF-CEM/R1 cells revealed an elongated marker chromosome containing a homogeneous staining region not present in the parent line. This chromosome appeared to be derived from chromosome 21.