We have isolated a genomic DNA fragment from HeLa cells containing the promoter region and the first two exons of the human gene encoding DNA topoisomerase I (hTOP1). Transcription of hTOP1 mRNA initiates at multiple sites which are clustered 247 nucleotides and 210 nucleotides upstream of the translation-initiation site of the protein coding region. The nucleotide sequence of the region preceding the transcription-initiation sites is G/C rich and contains sequence motifs which are known binding sites of the transcription factors Oct1 (octameric transcription factor 1), Sp1 and AP2 (activator protein 2). Furthermore, one cAMP-responsive element is present 50 nucleotides upstream of the transcription-initiation site nearest the 5' end. Neither TATA nor CAAT boxes were found in the promoter region of the hTOP1 gene. A 918-bp fragment containing the sequence elements described above drives the transient expression of a chloramphenicol acetyl transferase (CAT) gene sequence in transfected HeLa and 293 cells. In addition we analyzed a 10-kb fragment containing the promoter and exons 1 and 2 for regions of DNase I hypersensitivity. We detected one prominent DNase-I-hypersensitive region in the promoter close to the putative transcription-factor-binding sites and several weaker regions in intron 2.