Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfate conjugation of DHEA and other steroids. From 20 to 25% of subjects are included in a subgroup with high levels of hepatic DHEA ST activity, raising the possibility that this enzyme activity might be controlled by a genetic polymorphism. To understand the molecular mechanisms involved in regulating levels of DHEA ST activity in human tissue, we cloned the human DHEA ST gene, STD. STD spans at least 17 kb and is composed of 6 exons and 5 introns. The locations of the splice junctions for several of the introns are identical to those present in the rat phenol or aryl ST gene, the only other cytosolic ST gene for which the entire exon/intron structure has been reported, as well as those present in two partially characterized genes for the rat senescence marker protein, genes that are also thought to encode ST enzymes. The 5'-flanking region of the human STD gene does not contain canonical TATA or CCAAT elements, but this region is capable of promoting transcription of a reporter gene in Hep G2 cells. Molecular cloning and structural characterization of the human STD gene will make it possible to study genetic mechanisms involved in the regulation of DHEA ST activity in human tissue.