The lacdiNAc sequence GalNAc beta 1-->4GlcNAc beta 1-R occurs in the N- and O-glycans of many glycoproteins in vertebrate and invertebrates. We now report that both human 293 cells and Chinese hamster ovary (CHO) cells contain a UDPGalNAc:GlcNAc beta 1,4 N-acetylgalactosaminyltransferase (beta 1,4GalNAcT) that forms the lacdiNAc sequence. The beta 1,4GalNAcT in CHO cells is distinct from beta 1,4 galactosyltransferase in that the latter enzyme, but not the former, binds to a column of immobilized bovine alpha-lactalbumin. To determine whether endogenous glycoproteins in these cells contain lacdiNAc sequences, glycoproteins from 293 cells, CHO and Lec8 CHO cells were desialylated and passed over immobilized Wisteria floribunda agglutinin (WFA), a plant lectin with affinity for terminal GalNAc residues. WFA bound to approximately 120 and approximately 80 kDa glycoproteins in 293 cells and glycans from these glycoproteins contained lacdiNAc sequences. The approximately 120 kDa glycoproteins in 293 cells bound by WFA is a mixture of both the lysosome-associated membrane glycoproteins LAMPs-1 and -2. WFA bound to two glycoproteins of approximately 47 and approximately 78 kDa in Lec8 CHO cells, but these glycoproteins are not LAMPs and they do not contain the lacdiNAc sequence. Instead, they contain multiple GalNAc alpha-Ser/Thr O-glycans that promote binding to WFA. Thus, the beta 1,4GalNAcT in 293 cells displays a limited specificity in its recognition of acceptors, whereas the beta 1,4GalNAcT in CHO cells fails to promote synthesis of the cognate lacdiNAc sequence. The presence of the beta 1,4GalNAcT may not be sufficient for synthesis of lacdiNAc sequences and other factors may contribute to regulate the functionality of the enzyme.