A survey was made of the density of the cholinergic innervation of different parts of the brainstem of the rat and ferret. Sections of rat and ferret brainstems were stained for choline acetyltransferase (ChAT) immunoreactivity by using a sensitive immunocytochemical method. Adjacent sections were stained for acetylcholinesterase activity or Nissl substance. The density of the distribution of fine calibre, varicose ChAT‐positive axons, assumed to represent cholinergic terminals, was categorised arbitrarily into high, medium, or low. A high density of ChAT‐positive terminals was found in all or parts of these structures: interpeduncular nucleus, superficial grey layer of the superior colliculus (ferret), intermediate layers of the superior colliculus, lateral part of the central grey (rat), an area medial to the parabigeminal nucleus (rat), pontine nuclei, ventral tegmental nucleus (rat), midline pontine reticular formation, and an area ventral to the exit point of the 5th nerve (ferret). A medium density of ChAT‐positive terminals was observed in all or parts of: the substantia nigra zona compacta (ferret), ventral tegmental area (ferret), superficial grey layer of the superior colliculus, intermediate and deep layers of the superior colliculus, lateral central grey, area medial to the parabigeminal nucleus, inferior colliculus, dorsal tegmental nucleus, ventral tegmental nucleus (ferret), pontine nuclei, ventral nucleus of the lateral lemniscus (ferret), midline pontine reticular formation, ventral cochlear nucleus, dorsal cochlear nucleus, lateral superior olive, spinal trigeminal nuclei, prepositus hypoglossal nucleus, lateral reticular nucleus, paragigantocellular nucleus, and the dorsal column nuclei including the cuneate, external cuneate, and gracile nuclei. A low density of ChAT‐positive terminals was seen throughout the remainder of the brainstem of the rat and ferret, but these terminals were absent from the medial superior olive, substantia nigra zona reticulata (rat), and the central part of the ferret lateral superior olive. A pericellular‐like distribution of ChAT‐positive terminals was observed in the ventral cochlear nucleus and in association with some of the cells of the nucleus of the mesencephalic tract of the trigeminal nerve. A climbing fibre type arrangement of ChAT‐positive terminals was found in the substantia nigra zona compacta (ferret) and medial reticular formation. In general, the distribution of staining for AChE activity reflected that of the distribution of ChAT immunoreactivity in the brainstem, except in a few regions where there were also species differences in the distribution of ChAT‐positive terminals, e.g., in the superficial grey layer of the superior colliculus and in the substantia nigra.

It was concluded from this study that acetylcholine has an important role to play in the brainstem, especially in the sensory areas. The observation that the distribution of cholinergic fibres is mostly consistent between the rat and ferret suggests that brainstem cholinergic pathways have a similar organisation in diverse species. There does not, however, appear to be a point‐to‐point correspondence in the brainstem between the distribution of cholinergic terminals and of acetylcholine receptors (as determined by previous pharmacological studies).