To gain insight into neuromodulatory role of cholinergic signaling in different brainstem regions, we dialyzed atropine (50mM), a non‐specific muscarinic receptor blocker, into the brainstems of chronically cannulated goats. Atropine dialysis into the pontine Kölliker‐Fuse nucleus (KFN) at night decreased breathing (J. Appl. Physiol. 109: 159‐170, 2010). Atropine dialysis into the medullary pre‐Bötzinger complex (preBötC) increased breathing both at night and during the day (J. Appl. Physiol 114: 694‐704, 2013) with increases observed in substance P and glycine in the effluent mock cerebral spinal fluid (mCSF). Herein we report levels of neurochemicals in effluent mCSF during 50mM atropine dialysis into the KFN and lateral and medial parabrachial nuclei. There were no pontine site‐related differences in levels of tested neurochemicals; thus the data were pooled. In all pontine cannulated goats, during the day and at night, atropine dialysis increased substance P to the same level as atropine dialysis into the medulla (preBötC). In contrast, atropine dialysis into the pontine nuclei did not change levels of glutamine, glycine, or GABA in the effluent mCSF. These findings suggest that the site specific differences in the respiratory response to atropine dialysis are not due to alterations in substance P levels.Grant Funding Source: Supported by NIH HL007852, NIH HL25739, NIH HL112996, and the Department of Veteran Affairs