Dehydroepiandrosterone sulfotransferase catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundant steroid in circulation in the human and primate. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in peripheral target tissues. In addition, blockade at the dehydroepiandrosterone level could give raise to high level of DHEA and thus disorders due to mild excess of androgen. Recently, the cDNA encoding dehydroepiandrosterone sulfotransferase has been isolated from a human liver cDNA library. To study the regulation and expression, as well as the possible defect linked to DHEA sulfotransferase gene, we have isolated and characterized its structure by screening a lambda EMBL3 library of human leukocyte genomic DNA using human dehydroepiandrosterone sulfotransferase cDNA as a probe. Sequencing of the gene shows that it is included in approximately 17 kb and contains six exons separated by five introns. Northern blot analysis shows a strong signal in the adrenals and liver, whereas no signal was detected in the spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, lung, skeletal muscle, kidney, or pancreas. Using primer extension analysis, the transcription start site is located at nucleotide 98 upstream from the ATG initiating codon. Putative TATA and CAAT boxes are situated at positions 72 and 96 upstream from the transcription start site, respectively. Using DNA from a panel of human/rodent somatic cell hybrids, and amplification of the gene by the polymerase chain reaction, the human dehydroepiandrosterone sulfotransferase gene has been assigned to chromosome 19.