By employing reverse transcription-polymerase chain reaction (RT-PCR) in conjunction with 5'-rapid amplification of cDNA ends technique, we have cloned a novel mouse sulfotransferase cDNA. Database search led to the identification of a human gene encoding the homologue of this newly discovered mouse sulfotransferase. RT-PCR technique was employed to clone the cDNA encoding the human enzyme. Sequence analysis revealed that the novel mouse and human sulfotransferases display nearly 98% identity in their amino acid sequences. Their amino acid sequence identity to other known cytosolic sulfotransferases, however, was found to be below 36%. These two highly conserved sulfotransferases therefore appear to belong to a family different from the two major mammalian cytosolic sulfotransferase gene families. Northern blot analysis revealed the neuronal tissue-specific expression of these two novel sulfotransferases. Recombinant mouse and human brain sulfotransferases, expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as 33 kD proteins upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified mouse and human brain sulfotransferases displayed enzymatic activities toward endogenous and xenobiotic compounds, including L-triiodothyronine, thyroxine, estrone, p-nitrophenol, 2-naphthylamine, and 2-naphthol. Using mouse brain filtrate as substrate, both brain sulfotransferases were shown to catalyze specifically the sulfation of only a few compounds.