In neonatal rhythmic medullary slices, muscarinic acetylcholine receptor (mAChR) activation of hypoglossal (XII) motoneurons that innervate the tongue has a net excitatory effect on XII inspiratory motor output. Conversely, during rapid eye movement sleep in adult rodents, XII motoneurons experience a loss of excitability partly due to activation of mAChRs. This may be mediated by activation of G-protein-coupled inwardly rectifying potassium (GIRK) channels. Therefore, this study was designed to evaluate whether muscarinic modulation of XII inspiratory motor output in mouse rhythmic medullary slices includes GIRK channel-mediated inhibition and, if so, when this inhibitory mechanism emerges. Local pressure injection of the mAChR agonist muscarine potentiated inspiratory bursting by 150 ± 28% in ()- rhythmic medullary slice preparations. In the absence of muscarine, pharmacological GIRK channel block by Tertiapin-Q did not affect inspiratory burst parameters, whereas activation with ML297 decreased inspiratory burst area. Blocking GIRK channels by local preapplication of Tertiapin-Q revealed a developmental change in muscarinic modulation of inspiratory bursting. In - rhythmic medullary slices, Tertiapin-Q preapplication had no significant effect on muscarinic potentiation of inspiratory bursting (a negligible 6% decrease). However, preapplication of Tertiapin-Q to - rhythmic medullary slices caused a 19% increase in muscarinic potentiation of XII inspiratory burst amplitude. Immunofluorescence experiments revealed expression of GIRK 1 and 2 subunits and M1, M2, M3, and M5 mAChRs from to . Overall, these data support that mechanisms underlying muscarinic modulation of inspiratory burst activity change postnatally and that potent GIRK-mediated inhibition described in adults emerges early in postnatal life. Muscarinic modulation of inspiratory bursting at hypoglossal motoneurons has a net excitatory effect in neonatal rhythmic medullary slice preparations and a net inhibitory effect in adult animals. We demonstrate that muscarinic modulation of inspiratory bursting undergoes maturational changes from to that include emergence of an inhibitory component mediated by G-protein-coupled inwardly rectifying potassium channels after in neonatal mouse rhythmic medullary slice preparations.