In an attempt to identify cholinergic neurons in the brain stem which project to the spinal cord, [3H]choline (100, 20, 10, 5 or 1 microCi) was injected into the upper cervical spinal cord in 55 rats. The animals were killed 20 h later and the brains processed for autoradiography of diffusible substances. At all doses of [3H]choline, cells were consistently, retrogradely labeled in the medical medullary reticular formation, the lateral vestibular nucleus, the dorsolateral pontine tegmentum and the red nucleus. The retrogradely labeled cells were found to be moderately to darkly stained for acetylcholinesterase. Injection of [3H]noradrenaline (50 microCi) into the upper cervical spinal cord resulted in retrograde labeling of cells in the locus coeruleus, subcoeruleus and the ventrolateral pontine tegmentum, that correspond in position to the neurons of the A6, A7 and A5 catecholamine cell groups, respectively. Injection of [3H]serotonin (20 microCi) into the upper cervical spinal cord was associated with retrograde labeling of cells in the raphe pallidus, obscurus and magnus nuclei that correspond in position to those of the B1, B2 and B3 serotonin cell groups, respectively. Injection of True Blue into the upper cervical spinal cord was followed by retrograde labeling of a large number of cells located in the areas where cells were retrogradely labeled by [3H]choline, [3H]noradrenaline and [3H]serotonin, and additionally, in the solitary tract nucleus, the lateral, parvicellular medullary reticular formation, the caudal and oral pontine reticular formation, the mesencephalic reticular formation and the superior colliculus. These results indicate that from the cervical spinal cord, [3H]choline selectively retrogradely labels a certain population of non-monaminergic, acetylcholinesterase-positive cells localized in the medial medullary, and secondarily the dorsolateral pontine, reticular formation, the lateral vestibular nucleus, and the red nucleus.