We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated GalNAc-4-ST2 (GenBank TM accession number AF332472) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 443 amino acids with a 12-amino acid cytoplasmic domain, a 23-amino acid transmembrane domain, and a 408-amino acid luminal domain containing four potential N-linked glycosylation sites. GalNAc-4-ST2 displays a high degree of amino acid sequence identity with GalNAc-4-ST1 (46%), HNK-1 ST (23%), chondroitin 4-O-sulfotransferase-1 (C4ST-1) (27%), and chondroitin 4-O-sulfotransferase-2 (C4ST-2) (24%). GalNAc-4-ST2 transfers sulfate to the C-4 hydroxyl of terminal β1,4-linked GalNAc in the sequence GalNAc-β1,4GlcNAcβ-R found on N-linked oligosaccharides and nonterminal β1,4-linked GalNAc in chondroitin and dermatan. The translated region of GalNAc-4-ST2 is encoded by five exons located on human chromosome 18q11.2. Northern blot analysis reveals a 2.1-kilobase transcript. GalNAc-4-ST2 message is most highly expressed in trachea and to a lesser extent in heart, liver, pancreas, salivary gland, and testis. The I.M.A.G.E. cDNA clone 49547 contains a putative GalNAc-4-ST2 splice form with an open reading frame encoding a protein of 358 amino acids that lacks the transmembrane domain and the stem region. This form of GalNAc-4-ST2 is not retained by transfected cells and is active against chondroitin but not terminal β1,4-linked GalNAc. Thus, as with GalNAc-4-ST1, sequences N-terminal to the catalytic domain contribute to the specificity of GalNAc-4-ST2 toward terminal β1,4-linked GalNAc.