The tetrameric form of native serum-derived bovine acetylcholinesterase is retained in the circulation for much longer periods (mean residence time, MRT = 1390 min) than recombinant bovine acetylcholinesterase (rBoAChE) produced in the HEK-293 cell system (MRT = 57 min). Extensive matrix-assisted laser desorption ionization-time of flight analyses established that the basic structures of the N-glycans associated with the native and recombinant enzymes are similar (the major species (50-60%) are of the biantennary fucosylated type and 20-30% are of the triantennary type), yet the glycan termini of the native enzyme are mostly capped with sialic acid (82%) and alpha-galactose (12%), whereas glycans of the recombinant enzyme exhibit a high level of exposed beta-galactose residues (50%) and a lack of alpha-galactose. Glycan termini of both fetal bovine serum and rBoAChE were altered in vitro using exoglycosidases and sialyltransferase or in vivo by a HEK-293 cell line developed specifically to allow efficient sialic acid capping of beta-galactose-exposed termini. In addition, the dimeric and monomeric forms of rBoAChE were quantitatively converted to tetramers by complexation with a synthetic peptide representing the human ColQ-derived proline-rich attachment domain. Thus by controlling both the level and nature of N-glycan capping and subunit assembly, we generated and characterized 9 distinct bovine AChE glycoforms displaying a 400-fold difference in their circulatory lifetimes (MRT = 3.5-1390 min). This revealed some general rules and a hierarchy of post-translation factors determining the circulatory profile of glycoproteins. Accordingly, an rBoAChE was generated that displayed a circulatory profile indistinguishable from the native form.