Growing interest in cholinergic neuronal systems has caused a resurgence of thiocholine histochemical studies. Reports from different laboratories refer to "modified" Koelle-Friedenwald procedures for the histochemical localization of acetylcholinesterase (AChE) but the modifications are seldom specified. A glance at the literature suggests that there is considerable variability in results obtained in similar brain structures. We report a detailed thiocholine protocol and observations on several experimental factors which affect the sensitivity, consistency and the discreteness of localization of AChE in histological material. Attention to these factors and adherence to the protocol described here has yielded consistent results in several species, and in pre- and post-natal material. The value of AChE staining in conjunction with immunocytochemical localization of cholineacetyltransferase to study cholinergic neural structures is discussed.