We have isolated genomic DNA clones covering the gene for human DNA polymerase delta catalytic subunit (POLD1) and its 5' flanking sequence. This gene is divided into 27 exons and is distributed over at least 32 kb of DNA. The exons and most of the introns are relatively small. The sizes of the exons range from 55 to 201 bp. Seven introns are smaller than 100 bp. Intron 1 is the largest intron, with a size of greater than 10 kb. All of the intron-exon junctions match well with the reported consensus sequences. Multiple copies of the Alu repetitive sequence and the variable number of tandem repeats were found in several introns. Transcription of POLD1 appears to initiate at multiple sites. The major start site was 53 nucleotides upstream of the ATG start codon. The sequence of the promoter and upstream DNA is G+C rich and does not contain a TATA sequence. Several potential transcription factor-binding sites, including the AP2-, CTF-, Ets1-, GCF-, MBF-1-, NF-E1-, and Sp1-binding sites, were found in this region. A 1.8-kb pol delta promoter DNA directed the expression of a luciferase reporter gene when transfected into HeLa cells.