The paradox of high substrate turnover occurring within the confines of a deep, narrow gorge through which acetylcholine must traverse to reach the catalytic site of acetylcholinesterase has suggested the existence of transient gorge enlargements that would enhance substrate accessibility. To establish a foundation for the experimental study of transient fluctuations in structure, site-directed labeling in conjunction with time-resolved fluorescence anisotropy were utilized to assess the possible involvement of the omega loop (Omega loop), a segment that forms the outer wall of the gorge. Specifically, the flexibility of three residues (L76C, E81C, and E84C) in the Cys69-Cys96 Omega loop and one residue (Y124C) across the gorge from the Omega loop were studied in the absence and presence of two inhibitors of different size, fasciculin and huperzine. Additionally, to validate the approach molecular dynamics was employed to simulate anisotropy decay of the side chains. The results show that the Omega loop residues are significantly more mobile than the non-loop residue facing the interior of the gorge. Moreover, fasciculin, which binds at the mouth of the gorge, well removed from the active site, decreases the mobility of 5-((((2-acetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Huperzine, which binds at the base of active-site gorge, has no effect on the mobility of reporter groups attached to L76C and Y124C but increases the mobility of the reporter groups attached to E81C and E84C. Besides showing that fluctuations of the Omega loop residues are not tightly coupled, the results indicate that residues in the Omega loop exhibit distinctive conformational fluctuations and therefore are likely to contribute to transient gorge enlargements in the non-liganded enzyme.