Acetylcholinesterase (E.C.3.1.1.7) was partially purified from rat brains stored in toluene. Extraction was performed using buffers containing non-ionic tensoactive detergents. Some properties of the enzyme were affected by the use of different activity measurement methods, such as the short-time radiometric or the long-time colorimetric method. There were two zones of maximum activity in the range pH 7.5-8.0 and 8.0-8.6, respectively. There seems to be a histidine residue in the enzyme that participates in the catalytic process. Thermal denuration presented first order kinetics and different thermodynamic parameters were obtained on using different incubation periods. On using the short-time activity measurement method there was activation at high substrate concentration, but with the long time method there was a marked inhibition produced by excess of substrate. However, if the enzyme was extracted from fresh rat brain, toluene untreated, these differences dissapeared. Gel filtration and disc electrophoresis showed the presence of multiple and interconvertible forms of the enzyme.