Secreted human alkaline phosphatase (SEAP, a model protein containing a single N-glycan chain) was expressed in Spodoptera frugiperda Sf-9 (Sf-9) and Trichophisia ni BTI-Tn-5B1-4 (Tn-5B1-4) insect cell Lines infected with recombinant Autographa californica multiple nuclear polyhedrovirus expressing SEAP under control of the polyhedrin promoter. SDS-PAGE showed that both systems expressed fairly pure rSEAP products. The rSEAP expression level was 7.0 U/mL in Tn-5B1-4, higher than the 4.1 U/ml, produced by Sf-9. Kinetic analysis showed that V-max and K-m of human placental SEAP were approx 10-fold higher than that of rSEAP, whereas the V-max and K-m of rSEAP prepared using both insect cell lines were comparable. To characterize the recombinant SEAP (rSEAP) glycosylation, the purified rSEAP was digested with PNGase F to release the N-glycan chains. Glycan analysis showed the presence of oligomannose-type N-linked glycans (i.e., Man(2-8)GlcNAc(2) and FucMan(3or4)GIcNAc(2)) in rSEAP from Sf9 and Tn-5B1-4 cell lines. The p roportions of these oligosaccharide structures were different in the two cell lines. Man(4)GlcNAc(2) and FucMan(4)GlcNAc(2). were the major rSEAP N-glycans produced in Sf-9 cells, while Man(2)GlcNAc(2). was the major rSEAP N-glycan produced in Tn-5B1-4 cells.