Based on sequence homology with the previously cloned human cerebroside sulfotransferase (CST) cDNA, a novel sulfotransferase was cloned by screening a human fetal brain cDNA library. The novel sulfotransferase gene was present on human chromosome 11q13; the location was different from human CST and from that of the recently cloned human beta-Gal 3'-sulfotransferase (GP3ST). The isolated cDNA contained an open reading frame that encoded a predicted protein of 431 amino acid residues with type II transmembrane topology. The amino acid sequence showed 33% identity with that of human CST and 38% with that of human GP3ST. The recombinant enzyme expressed in Chinese hamster ovary cells catalyzed transfer of sulfate to position 3 of non-reducing beta-galactosyl residues in Galbeta1-4GlcNAc. Type 2 chains served as good acceptors, whereas type 1 chains served as poor acceptors, and intermediate activity was found toward Galbeta1-3GalNAc. Therefore, the substrate specificity was different from that of GP3ST. CST activity was not detected in the newly cloned enzyme. Northern blotting analysis showed that the sulfotransferase mRNA was strongly expressed in the thyroid and moderately expressed in the brain, heart, kidney, and spinal cord. Co-transfection of the enzyme cDNA and fucosyltransferase III into COS-7 cells resulted in expression of (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc and a small amount of (SO(4)-3)Galbeta1-3(Fucalpha1-4)GlcNAc. These results indicated that the newly cloned enzyme is a novel Gal-3-O-sulfotransferase and is involved in biosynthesis of the (SO(4)-3)Galbeta1-4(Fucalpha1-3)GlcNAc structure.