The localization of cholinergic neurons in the cat lower brain stem was determined immunocytochemically with a monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. ChAT-positive neurons were observed in four major cell groups: cranial nerve motor and special visceromotor neurons: parasympathetic preganglionic visceromotor neurons; neurons located in the ponto-mesencephalic tegmentum including area X (or pedunculopontine tegmental nucleus), nucleus laterodorsalis tegmenti (Ldt) of Castaldi, and peri-locus coeruleus alpha (peri-alpha); and neurons located in nucleus reticularis magnocellularis (Mc) and adjacent nucleus reticularis gigantocellularis (Gc) of the medulla.