Sulfae conjugation is an important pathway in the metabolism of a large number of exogenous and endogenous compounds. These reactions are catalyzed by sulfotransferase (SULT) enzymes that utilize 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfate donor. PAPS is synthesized from ATP and inorganic sulfate by PAPS synthetase (PAPSS), Two separate PAPSS cDNAs, PAPSS1 and PAPSS2, have been identified in human tissues, We have cloned and characterized the genes for human PAPSS1 and PAPSS2 to make it possible to study the pharmacogenomics of these enzymes. Both genes consisted of 12 exons with virtually identical exon-intron splice junction locations. All splice junctions conformed to the "GT-AG" rule. The total length of PAPSS1 was approximately 108 kb, while that of PAPSS2 was greater than 37 kb. The 5'-flanking region of PAPSS1 did not include a TATA box sequence near the site of transcription initiation, but PAPSS2 had a TATA motif located 21 bp upstream from the site of transcription initiation. Northern blot analysis showed that the major PAPSS1 and PAPSS2 transcripts were approximately 2.7 and 4.2 kb in length, respectively, PAPSS1 mapped to human chromosome band 4q24 while PAPSS2 mapped to 10q22-23 by fluorescence in situ hybridization analysis. Cloning and structural characterization of PAPSS1 and PAPSS2 will make it possible to perform molecular genetic and pharmacogenomic studies of these important enzymes in humans.