A limited number of glycoproteins including luteinizing hormone (LH) and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with 1,4-linked GalNAc. The selective addition of GalNAc to these glycoproteins requires that the 1,4-N-acetylgalactosaminyltransferase (GT) recognize both the oligosaccharide acceptor and a peptide-recognition determinant on the substrate glycoprotein. We report here that two recently cloned GTs, GT3 and GT4, that are able to transfer GalNAc to GlcNAc in 1,4-linkage display the necessary glycoprotein specificity in vivo. Both GTs transfer GalNAc to N-linked oligosaccharides on the LH alpha subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxy terminal 19 amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. Addition of this 19 amino acid sequence to the carboxy terminus of Trf confers full acceptor activity onto Trf for both GT3 and GT4 in vivo. The complete 19 amino acid sequence is required for optimal GalNAc addition in vivo, indicating that the peptide sequence is both necessary and sufficient for recognition by GT3 and GT4.