The GalNAc beta 1,4GlcNAc (LacdiNAc or LDN) structure is a more common structural feature in invertebrate glycoconjugates when compared with the Ga1 beta 1,4GlcNAc structure. Recently, beta 1,4-N-acetylgalactosaminyltransferase (beta 4GalNAcT) was identified in some invertebrates including Drosophila. However, the LDN structure has not been reported in Drosophila, and the biological function of LDN remains to be determined. In this study, we examined acceptor substrate specificity of Drosophila beta 4GalNAcTA by using some N- and O-glycans on glycoproteins and neutral glycosphingolipids (GSLs). GalNAc was efficiently transferred toward N-glycans, O-glycans, and the arthro-series GSLs. Moreover, we showed that d beta 4GalNAcTA contributed to the synthesis of the LDN structure in vivo. The d beta 4GalNAcTA mRNA was highly expressed in the developmental and adult neuronal tissues. Thus, these results suggest that d beta 4GalNAcTA acts on the terminal GlcNAc residue of some glycans for the synthesis of LDN, and the LDN structure may play a role in the physiological or neuronal development of Drosophila. (c) 2007 Elsevier Inc. All rights reserved.