Human dihydrofolate reductase gene is located in chromosome 5 and is unlinked to the related pseudogenes.

The chromosomal location of the human dihydrofolate reductase (DHFR; EC 1.5.1.3) gene that is amplified in a methotrexate-resistant human cell line has been investigated by screening a large number of human-mouse cell hybrids containing overlapping subsets of human chromosomes. A correlation of genomic blotting data with the chromosome constitution of the individual cell hybrids has allowed the assignment of the human DHFR gene to chromosome 5. This chromosome assignment has been confirmed by the observation of a concomitant loss of the human DHFR gene and of sensitivity to diphtheria toxin, a marker associated with chromosome 5, in two human-mouse cell hybrids selected for resistance to the toxin. Six EcoRI fragments of human DNA containing DHFR pseudogenes or other DHFR-related sequences have been assigned to chromosomes other than chromosome 5.