A novel human chondroitin 6-O-sulfotransferase, designated C6ST-2, was identified by BLAST analysis of expressed sequence tag using the sequence of a previously described human chondroitin 6-O-sulfotransferase (C6ST-1) as a probe. The new cDNA sequence revealed an open reading frame coding for a protein of 486 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 24% identity to the human C6ST-1, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active sulfotransferase with marked specificity for polymer chondroitin. In contrast, keratan sulfate and oligosaccharides containing the Galbeta1-4GlcNAc sequence, which are good acceptor substrates for the C6ST-1, hardly served as acceptors. The identification of the reaction product indicated that the enzyme is a novel chondroitin 6-O-sulfotransferase (C6ST-2) that mainly transfers sulfate to N-acetylgalactosamine. The coding region of C6ST-2 was contained in a single exon and localized to chromosome Xp11. Northern blot analysis of human brain poly(A)(+) RNA revealed a single transcript of 2.4 kilobase pairs. Reverse transcription-polymerase chain reaction analysis showed that C6ST-2 is developmentally regulated in various tissues with expression persisting through adulthood in the spleen. Thus, we demonstrated the redundancy in chondroitin 6-O-sulfotransferases capable of forming chondroitin 6-sulfate, which is important for understanding the mechanisms leading to specific changes in the sulfation profile of chondroitin sulfate chains in various tissues during development and malignant transformation.