The distribution of acetylcholine neurons in the brainstem of the cat was studied by choline acetyltransferase (ChAT) immunohistochemistry and compared to that of catecholamine neurons examined in the same or adjacent sections by tyrosine hydroxylase (TH) immunohistochemistry. The largest group of ChAT-positive (+) neurons was located in the lateral pontomesencephalic tegmentum within the pedunculopontine tegmental nucleus and the laterodorsal tegmental nucleus rostrally and within the parabrachial nuclei and locus coeruleus nucleus more caudally. TH+ neurons were found to be coextensive and intermingled with ChAT+ neurons in the dorsolateral pontomesencephalic tegmentum, where the number of ChAT+ cells (approximately 18,500) exceeded that of the TH+ cells (approximately 12,000). In the caudal pons, scattered ChAT+ neurons were situated in the ventrolateral tegmentum together with TH+ neurons. In the medulla, numerous ChAT+ cells were located in the lateral tegmental field, where they extended in a radial column from the dorsal motor nucleus of the vagus to the ventrolateral tegmentum around the facial and ambiguus nuclei, occupying the position of preganglionic parasympathetic neurons of the 7th, 9th, and 10th cranial nerves. TH+ cells were also present in this field. Neurons within the general visceral, special visceral, and somatic motor cranial nerve nuclei were all immunoreactive to ChAT. Scattered ChAT+ neurons were also present within the medullary gigantocellular and magnocellular tegmental fields together with a small number of TH+ neurons. Other groups of ChAT+ cells were identified within the periolivary nuclei, parabigeminal nucleus, prepositus hypoglossi nucleus, and the medial and inferior vestibular nuclei. Acetylcholine neurons thus constitute a heterogeneous population of cells in the brainstem, which in addition to including the somatic and visceral efferent systems, comprises many other discrete systems and represents an important component of the brainstem reticular formation. The proximity to and interdigitation with catecholamine neurons within these systems may be of important functional significance.