The earliest studies on the regional distribution of acetylcholinesterase (AChE) within the central nervous system were based on the determination of the amount of CO liberated by homogenates of selected areas in the presence of an ester of 2 choline and a bicarbonate buffer. Using this biochemical approach, Burgen and Chipman (1951) were able to establish that acetylcholinesterase is not evenly distributed within the central nervous system. They found that the cerebellum, the lateral geniculate body, and the striatum contained a high concentration of AChE. The high concentration of AChE in the striatum could be correlated with a higher rate of acetylcholine synthesis. However, this was not the case for the cerebellum, where acetylcholine synthesis was very low. Other in vitro studies have been aimed at establishing the regional distribution of the other two components of the cholinergic system, cholinacetylase (ChA) and acetylcholine (ACh). An equally asymetrical distribution for these substances has been established in vitro (MacIntosh, 1941 ; Feldberg and Mann, 1946; Feldberg and V ogt, 1948; MacIntosh and Oborin, 1953; Quastel, 1962; Mitchell, 1963; Krnjevic and Phillis, 1963; Aprison et al., 1964; McLennan, 1964; Cohen, 1956). The in vitro determination of acetylcholinesterase (Koelle, 1950; Burgen and Chipman, 1951; Giacobini, 1959; Bennett et al., 1966; Fahn and Cote, 1968; Miller et al., 1969) presents the advan tage of permitting the use of a substrate like ACh which is a normally occurring ester of choline so that the establishment of enzyme specificity is less questionable.