Like lower and higher eucaryotes, insects have alpha 1,2-mannosidases which function in the processing of N-glycans. We previously cloned and characterized an insect alpha 1,2-mannosidase cDNA and demonstrated that it encodes a member of a family of N-glycan processing alpha 1,2-mannosidases (Kawar, Z., Herscovics, A., Jarvis, D.L., 1997. Isolation and characterisation of an alpha 1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9. Glycobiology 7, 433-443). These enzymes have similar protein sequences, require calcium for their activities, and are sensitive to 1-deoxymannojirimycin, but can have different substrate specificities and intracellular distributions. We recently determined the substrate specificity of the insect alpha 1,2-mannosidase, SfManI (Kawar, Z., Romero, P., Herscovics, A., Jarvis, D.L., 2000. N-glycan processing by a lepidopteran insect and 1,2-mannosidase. Glycobiology 10, 347-355). Now, we have examined the biosynthesis and subcellular localization of SfManI. We found that SfManI is partially N-glycosylated and that N-glycosylation is dramatically enhanced if the wild type sequon is changed to one that is highly utilized in a mammalian system. We also found that an SfManI-GFP fusion protein had a punctate cytoplasmic distribution in insect cells. Colocalization studies indicated that this fusion protein is localized in the Golgi apparatus, not in the endoplasmic reticulum or lysosomes. Finally, N-glycosylation had no influence over the substrate specificity or subcellular localization of SfManI.