This distribution of choline acetyltransferase (CHAT) immunoreactivity (CHAT-I) in the rat lower brain stem was analyzed using a highly sensitive avidin-biotin immunocytochemical method and 3-amino-9-ethyl-carbazole visualization. A much wider and more abundant distribution of CHAT-I structures in the lower brain stem was demonstrated than in earlier studies. The following areas were newly identified as areas rich in CHAT-I fibers: the interpeduncular nucleus, medial geniculate body, central gray matter of pons, pontine nucleus, parabigeminal nucleus, dorsal tegmental nucleus of Gudden, lateral trapezoid nucleus, inferior colliculus, dorsal and ventral cochlear nuclei, medial and lateral vestibular nuclei, reticular formation of medulla oblongata, and gelatinosa of caudal trigeminal spinal tract nucleus. In addition to the areas in which they have been known to exist, CHAT-I perikarya were found in the caudal portion of substantia nigra pars reticulata, the area between trigeminal motor nucleus and superior olivary nucleus, the medial and spinal vestibular nucleus, prepositus hypoglossal nucleus, raphe magnus and obscurus, ventromedial portion of solitary tract nucleus and its just ventral reticular formation, and caudal trigeminal spinal tract nucleus.