Molecular cloning, mapping, and DNA sequencing techniques have been used to characterize the functional human dihydrofolate reductase (DHFR) gene. The gene is about 30 kilobases in length. Its coding portions are separated into 6 exons, the intron-exon boundaries of which are identical to those of the previously characterized mouse DHFR gene. The 5 introns vary in length from 362 to 12,000 base pairs. The position of the DHFR gene promoter was identified as being shortly upstream from the initiation codon in an in vitro transcription reaction by polymerase II. A DHFR minigene was constructed in a plasmid expression vector by combining a DNA fragment containing exon 1, intron I, and a small part of exon 2 from the functional gene, with a second DNA fragment containing exons 2-6 from a processed intronless gene, the coding sequences of which are identical to those of the normal locus. Transcription initiation from the DHFR promoter was localized to a position 71 +/- 2 base pairs upstream from the initiation codon, both in monkey kidney cells transfected with vectors containing the DHFR minigene, and in human HeLa cells. This single transcription start and the three previously identified polyadenylation sites account for the 800-, 1,000-, and 3,800-nucleotide DHFR mRNA species found in human cells. On comparison of the mouse and human DHFR genes, sequence homology was shown to be limited to the coding regions and 100 base pairs of the 3' untranslated region up to the first polyadenylation site of both genes. In addition, there is fairly extensive homology in the 5' flanking region, although the quadruply repeated 48-base pair sequence found in the mouse genome is represented only once in human DNA.