We have cloned two GalNAc-4-sulfotransferases, GalNAc-4-ST1 and GalNAc-4-ST2, that transfer sulfate to terminal β1,4-linked GalNAc. In conjunction with the action of protein-specific β1,4GalNAc-transferases, GalNAc-4-ST1 and GalNAc-4-ST2 account for the presence of terminal β1,4-linked GalNAc-4-SO₄ on glycoproteins such as lutropin, thyrotropin (TSH), proopiomelanocortin (POMC), carbonic anhydratase-VI (CA-VI), and tenascin-R. GalNAc-4-ST1 and GalNAc-4-ST2 can be distinguished by their differing specificity for oligosaccharide acceptors and temperature lability. The differences in properties have been used to show that the levels of GalNAc-4-ST1 and GalNAc-4-ST2 activity are proportionate to the levels of their respective transcripts. Furthermore, we have found that both transcript and activity levels of GalNAc-4-ST1 and GalNAc-4-ST2 vary widely among different tissues indicating that the regulation of their expression differs. Differences in specificity and the regulation of expression may account for existence of two GalNAc-4-sulfotransferases in vivo. The highest levels of both GalNAc-4-ST1 and GalNAc-4-ST2 transcripts are present in the pituitary of the mouse with multiple cell types that produce glycoproteins terminating with GalNAc-4-SO₄. Genetic ablation of both GalNAc-4-ST1 and GalNAc-4-ST2 may be necessary to alter the pattern and/or extent of sulfate addition to terminal β1,4GalNAc in tissues such as pituitary.