A limited number of glycoproteins including luteinizing hormone and carbonic anhydrase-VI (CA6) bear N-linked oligosaccharides that are modified with β1,4-linked N-acetylgalactosamine (GalNAc). The selective addition of GalNAc to these glycoproteins requires that the β1,4-N- acetylgalactosaminyl-transferase (βGT) recognize both the oligosaccharide acceptor and a peptide recognition determinant on the substrate glycoprotein. We report here that two recently cloned βGTs, βGT3 and βGT4, that are able to transfer GalNAc to GlcNAc in β1,4-linkage display the necessary glycoprotein specificity in vivo. Both βGTs transfer GalNAc to N-linked oligosaccharides on the luteinizing hormone α subunit and CA6 but not to those on transferrin (Trf). A single peptide recognition determinant encoded in the carboxyl-terminal 19-amino acid sequence of bovine CA6 mediates transfer of GalNAc to each of its two N-linked oligosaccharides. The addition of this 19-amino acid sequence to the carboxyl terminus of Trf confers full acceptor activity onto Trf for both βGT3 and βGT4 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 βGT3 and βGT4.