Nucleotide sequences of two mouse cDNAs encoding new sulfotransferase enzymes were determined. Analysis of the deduced amino acid sequences revealed that one represents a novel member of the phenol sulfotransferase family and the other is highly homologous to human SULT2B1 hydroxysteroid sulfotransferases. The recombinant enzymes, transiently expressed in COS-7 cells, were characterized with respect to their substrate specificity using a variety of substrates for different types of sulfotransferases. The tissue-specific expression of these two new mouse sulfotransferases was examined by Northern blot analysis.