The cholinergic system plays an important role in brain homeostasis and interacts with the neuropeptidergic systems, and the functional relationships between both systems are well known. However, in the brainstem the possible physiological interactions between neurokinins and acetylcholine are unknown, although both substances have been detected in the same brainstem nuclei and have been implicated in similar functions controlled from brainstem regions such as some cranial motor nuclei. The aim of this work is to determine whether these possible physiological interactions might have a neuroanatomical basis by means of the double immunohistochemical detection of neurokinins (NK) and the enzyme choline acetyl-transferase (ChAT) in the human brainstem. No double-labelled structures were detected, although both NK and ChAT were observed in cell bodies and fibers of the same brainstem nuclei. The distribution of immunoreactive fibers is widespread, and NK and ChAT were observed in several motor cranial nerves as well as in the substantia nigra. The results obtained in the present work provide a neuroanatomical basis for possible physiological interactions between NK and ChAT that may be carried out by volume-transmission mechanisms. These interactions might participate in motor regulation or in limbic pathways as well as influence on other neurotransmitter systems such as the dopaminergic system. Copyright © 2019 Elsevier B.V. All rights reserved.