Schneider 2 (S2) cells from Drosophila melanogaster have been used as a plasmid-based, non-lytic expression system for foreign proteins. Here, a plasmid encoding the human erythropoietin (hEPO) gene fused with a hexahistidine (His(6)) tag under the control of the Drosophila metallothionein (MT) promoter was stably transfected into Drosophila S2 cells. After copper sulfate induction, transfected S2 cells were found to secrete hEPO with a maximum expression level of 18 mg/L and a secretion efficiency near 98%. The secreted hEPO from Drosophila S2 had an apparent molecular weight of about 23 similar to 27 kDa which was significantly lower than a recombinant hEPO expressed in Chinese hamster ovary (CHO) cells (about 36 kDa). N-glycosidase F digestion almost completely eliminated the difference and resulted in the same molecular weight (similar to 20 kDa) of de-N-glycosylated hEPO proteins. These data suggest that recombinant hEPO from S2 cells was modified with smaller N-glycans. Subsequently, the major N-glycans were identified following glycoamidase A digestion, labeling with 2-aminopyridine (PA), and two-dimensional high-performance liquid chromatography (HPLC) analysis in concert with exoglycosidase digestion. This analysis of N-glycans revealed that hEPO was modified to include paucimannosidic glycans containing two or three mannose residues with or without core fucose. A similar glycosylation pattern was observed on a recombinant human transferrin expressed in S2 cells. These results provide a detailed analysis of multiple N-glycan structures produced in a Drosophila cell line that will be useful in the subsequent application of these cells for the generation of heterologous glycoproteins. (c) 2005 Wiley Periodicals, Inc.