The St-20 and ST-40 cDNAs encode rat liver hydroxysteroid sulfotransferases (HS-ST) that are 90% identical in amino acid sequence but exhibit different substrate preferences for dehydroepiandrosterone (DHEA), androsterone (AD), and cortisol (CS). ST-40 is active for all three substrates, whereas ST-20 is mainly active for cortisol. To determine the domain responsible for the substrate preferences of the HS-STs, 20 chimeric HS-STs were constructed by reciprocal exchanges of DNA fragments derived from the cDNAs and were expressed in Escherichia coli. Some chimeric enzymes were enzymatically active for all three substrates, and some displayed reduced or lost CS-ST activity, with retention of DHEA- and AD-ST activities. Others lost all HS-ST activity. Analysis revealed that a central region (region III spanning amino acids 102-164 with five amino acid differences between ST-20 and ST-40) is essential for HS-ST activity, whereas regions II (amino acids 65-101) and IV (amino acids 165-219) are unimportant with regard to substrate preference. It was also shown that the parental combination of regions I (amino acids 1-64) and V (amino acids 220-284) is essential for CS-ST activity. Photoaffinity labeling with [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) revealed that some inactive chimeras lost affinity for PAPS. These results suggested that an ordered structure formed by regions I, III, and V is required for HS-ST activity, especially for substrate preference and PAPS binding.