A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a ß4-galactosyltransferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a ß4-galactosyltransferase family member was experimentally confirmed by expression and direct biochemical analyses. The newly identified ß4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as it was able to transfer galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three types of acceptors and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a ß4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on an in-depth analysis of the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan acceptor in vitro. The structure of the reaction product was confirmed by detailed chromatographic, mass spectroscopic, and nuclear magnetic resonance spectroscopic analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Finally, confocal microscopy showed that a GFP-tagged version of the enzyme was predominantly localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a ß4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and might function in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contributes to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.