The human cytosolic sulfotransferases (STs), dehydroepiandrosterone sulfotransferase (DHEA-ST) and the phenol-sulfating form of phenol sulfotransferase, (P-PST), have been expressed in bacteria and used to investigate the ability of the cloned enzymes to conjugate steroids and related compounds. DHEA-ST was capable of sulfating all of the 3-hydroxysteroids, testosterone and estrogens tested as substrates. The 3-hydroxysteroids, androsterone, epiandrosterone and androstenediol, were conjugated at 50-60% of the rate of DHEA. Of the steroids tested, P-PST was capable of conjugating only the estrogens. The catechol estrogens, 2-hydroxyestradiol, 4-hydroxyestradiol and 4-hydroxyestrone, and compounds with estrogenic activity such as 17 alpha-ethynyl-estradiol and trans-4-hydroxytamoxifen, were also tested as substrates. DHEA-ST showed little or no sulfation activity with these compounds; however, all of these compounds were sulfated by P-PST. These results indicate that the expressed human STs are valuable in analyzing the overlapping substrate specificities of these enzymes and that P-PST may have an important role in the metabolism of estrogens and estrogenic compounds in human tissues.