The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries. These subunits consisted of an equal number of amino acid residues with only one amino acid substitution. ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol. This strongly suggested that they are essential components of STa. The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.