In lamprey, brainstem reticulospinal (RS) neurons constitute the main descending input to the spinal cord and activate the spinal locomotor central pattern generators. Cholinergic nicotinic inputs activate RS cells and, consequently, induce locomotion. Cholinergic muscarinic agonists also induce locomotion when applied to the brainstem of birds. This study examined whether bath applications of muscarinic agonists could activate RS cells and initiate motor output in lampreys. Bath applications of 25µM muscarine elicited sustained, recurring depolarizations (mean duration of 5.0±0.5s recurring with a mean period of 55.5±10.3s) in intracellularly recorded rhombencephalic RS neurons. Calcium imaging experiments revealed that muscarine induced oscillations in calcium levels that occurred synchronously within the RS neuron population. Bath application of TTX abolished the muscarine effect, suggesting the sustained depolarizations in RS neurons are driven by other neurons. A series of lesion experiments suggested the caudal half of the rhombencephalon was necessary. Microinjections of muscarine (75µM) or the muscarinic receptor (mAchR) antagonist atropine (1mM) lateral to the rostral pole of the posterior rhombencephalic reticular nucleus induced or prevented, respectively, the muscarinic RS neuron response. Cells immunoreactive for muscarinic receptors were found in this region and could mediate this response. Bath application of glutamatergic antagonists (AP-5/CNQX) abolished the muscarine effect suggesting glutamatergic transmission is required for the effect. Ventral root recordings showed spinal motor output coincides with RS neuron sustained depolarizations. We propose that unilateral mAchR activation on specific cells in the caudal rhombencephalon activates a circuit that generates synchronous sustained, recurring depolarizations in bilateral populations of RS neurons.