Recently, cDNAs encoding human chondroitin 4-O-sulfotransferase-1 and -2 (C4ST-1 and C4ST-2) were cloned based on their similarity to HNK-1 sulfotransferase (HNK-1ST) (Hiraoka, N., Nakagawa, H., Ong, E., Akama, T.O., Fukuda, M.N., and Fukuda, M. [2000] Molecular cloning and expression of two distinct human chondroitin 4-O-sulfotransferasesthat belong to the HNK-1 sulfotransferase gene family. J. Biol. Chem., 275, 20188–20196). In the present study, we identified two additional novel sulfotransferases by searching the expression sequence tag and genomic DNA database for enzymes similar to C4ST-1 and C4ST-2. These newly cloned enzymes, termed GalNAc4ST-1 and GalNAc4ST-2, belong to the HNK-1ST gene family having 40–42% identity with C4ST-1. GalNAc4ST-1 and -2 do not add sulfate to HNK-1 precursor glycans, chondroitin, or desulfated dermatan sulfate. Instead, both enzymes can transfer sulfate to the 4-position of GalNAc in the context of GalNAcβ1→4GlcNAcβ1→R attached to both N-linked and core 2 branched O-linked oligosaccharides. GalNAc4ST-1 and -2 transcripts are highly expressed in the pituitary gland and trachea, respectively, and GalNAc4ST-1 and -2 transcripts are reciprocally expressed in other tissues as well. Moreover, both enzymes can transfer sulfate to lutropin, a pituitary glycoprotein hormone. These combined results indicate that GalNAc4ST-1 and -2 play critical roles in forming sulfo→4GalNAcβ1→4GlcNAcβ1→R in both N-glycans and O-glycans in a tissue-specific manner.