While the distribution of AChE in the central nervous system remains largely unexplained, neurons with very high levels of AChE are frequently identified as cholinergic, and cholinergic neurons always have high levels of AChE. This supports the utility of the empirical rule, that identification of cholinergic neurons requires demonstration of high levels of AChE. The application of this rule in conjuction with neuroanatomical and biochemical data has already provided substantial new insights into the organization of central cholinergic systems. It is evident that AChE histochemistry continues to be a valuable neurobiological tool, particularly in characterizing neurons that may be cholinergic, and also in identifying those neurons that cannot be cholinergic.