A lymphoblastic leukemia cell line, L5178YR, that is over 100,000-fold resistant to methotrexate (MTX) has been developed. Previous work has demonstrated that the resistant line has dihydrofolate reductase (DHFR) levels 300-fold higher than the sensitive parental cells, L5178Y. Resistant cells grown in the absence of MTX (L5178YR–) showed a similar level of DHFR activity. The increase in DHFR could be entirely accounted for by a corresponding increase in DHFR-specific mRNA and gene copies. Studies were carried out to determine whether these changes in the resistant cells were accompanied by karyotypic alterations. A detailed karyotypic analysis of the sensitive parental line (L5178YS) and the two resistant cell lines (L5178YR+ and L5178YR-) was performed. Although certain abnormal marker chromosomes were present in all three cell lines, the most striking consistent difference between the resistant and sensitive cells was the presence of a large, faintly banded region of intermediate staining intensity, termed a “homogeneously staining region” (HSR), inserted within a reduplicated part of chromosome 2. It was present in approximately 90% of resistant cells, and no more than one HSR was ever present in a cell. Hybridization in situ was performed to determine the chromosomal locations of DHFR genes. Utilizing a purified complementary DNA probe made from messenger RNA of the L5178YR+ cells, the genes were shown to be localized exclusively to th