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 number of human-Chinese hamster ovary cell hybrids containing terminal and interstitial deletions in human chromosome 5. A correlation of genomic blotting data with the chromosome 5 constitution of the individual hybrids has allowed the assignment of the human DHFR gene to 5q23. The present work also establishes the location of the related intronless pseudogene psi HD1 in chromosome 3.