Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647-658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans=200, 740, and 1055 min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.