The CNS of the tobacco hornworm, Manduca sexta, provides a rich source of true acetylcholinesterase (AChE, acetylcholine, hydrolase, EC 3.1.1.7). Optimal extraction of the enzyme was obtained with a nonionic detergent at high ionic strength (1% Triton X-100, 0.5 M NaCl). Velocity sedimentation of the Triton + salt-extracted enzyme demonstrated a single peak whose sedimentation coefficient was dependent upon the enzyme concentration layered on top of the gradient. When more than 20 units were applied to the gradient, a sedimentation coefficient of 8.6 S (205,000) was obtained, and extrapolation to zero units yielded a 5.7 S (110,500) species. Sedimentation in the absence of detergents (1.0 M NaCl or 10 mM phosphate buffer, pH 7.4) yielded pelleted enzyme and species with mean values of 18.6 S (650,000) and 17.5 S (600,000), respectively. The detergent-extracted enzyme also demonstrated a concentration-dependent size in gel filtration experiments. When less than 300 units were applied to the column, a single species was recovered, with a molecular radius of 40.15 +/- 2.08 A (108,000) or 43.4 +/- 2.38 A (117,000) calculated by different methods. If the sample contained 300 to 1,300 units, two species were observed, with molecular radii of 40.15 +/- 2.08 A or 43.4 +/- 2.38 A and 78.4 +/- 3.94 A (319,000) or 80.25 +/- 3.01 A (326,000). Velocity sedimentation and gel filtration of AChE have demonstrated that the enzyme has a minimum molecular weight of approximately 110,000 and also exists as higher-molecular-weight aggregates of this value.