Glycosylation is both cell line and protein dependent. Culture conditions can also influence the profile of glycoforms produced. To examine this possibility in the insect cell/baculovirus system, structures of N-linked oligosaccharides attached to SEAP (human secreted alkaline phosphatase), expressed under various culture conditions in BTI Tn5B1-4 cells, were characterized using FACE (fluorescence-assisted carbohydrate electrophoresis). Parameters varied were time of harvest, ammonia added during infection, dissolved oxygen, and temperature. It was found that glycosylation in the insect cell/ baculovirus expression system is a robust, stable system that is less perturbed by variations in culture conditions than the level of protein expression. Addition of ammonia and low oxygen conditions affected SEAP expression, but not the oligosaccharide profile of SEAP. Time of SEAP harvest increased the amount of ␣-mannosidase resistant structures from 4.1% at 34 hours postinfection (h pi), to 5.0% at 100 h pi, and to 7.5% at 120 h pi. These structures were primarily sensitive to Nacetylhexosaminidase digest, although a small amount was insensitive to both mannosidase and N-acetylhexosaminidase digests. Lowering the temperature from 28°C to 24°C or even 20°C, resulted in a twofold increase in oligosaccharides containing terminal ␣(1,3)-mannose residues. This condition did not affect the amount of mannosidase-resistant structures. However, this could result in more complete glycosylation of recombinant proteins in the BTI Tn5B1-4 cell line, because more structures with the potential for further processing would be produced.