1. 1.|Human erythrocyte acetylcholine hydrolase (EC 3.1.1.7) is purified 2537-fold, by isolating erythrocyte membrane fragments, by Triton X-100 extraction of the enzyme from the membranes and by DEAE-cellulose and calcium phosphate gel chromatographies. 2. 2.|The purified enzyme is a glycoprotein; it moves as a single band in acrylamide gel electrophoresis. Electron microscopy, gel filtration and density-gradient centrifugation indicate it to be a macromolecule, composed of small spherical units. The enzyme easily forms reversible aggregates and the presence of other proteins ( e.g. bovine serum albumin) preserve the purified enzyme, as a catalytically active polymer. 3. 3.|The molecular characteristics, the substrate specificity, the influence of pH and temperature on hydrolysis, and the kinetic constants of the enzyme are different from other cholinesterases implicated in the acetylcholine receptor function. 4. 4.|The kinetic characteristics suggest that the enzyme possesses allosteric behavior. The polar substrate, acetylthiocholine, activates the enzyme; a specifically low binding constant for the substrate binding, outside the active center, is calculated. Other quarternary nitrogen carrying compounds ( e.g. mytelase) are also found either to activate or inhibit the enzyme and compete with the substrate, depending upon the substrate concentration and hence upon the substrate activation of the enzyme. The kinetic behavior of the enzyme also depends upon the temperature, a highly ordered catalysis can only be observed above 32 °C; this result may also be explained by the allosteric behavior of the enzyme. 5. 5.|Specific anionic activators of the enzyme such as HCO 3 −, cis-oxaloacetate and fructose 1,6-diphosphate are established. Oxaloacetate binding to the enzyme is also found to be specific; this compound, similar to mytelase, is a better activator of the substrate activated enzyme.