Inspiratory hypoglossal motoneurons (IHMNs) maintain upper airway patency. However, this may be compromised during sleep and by sedatives, potent analgesics, and volatile anesthetics by either depression of excitatory or enhancement of inhibitory inputs. In vitro data suggest that serotonin (5-hydroxytryptamine, 5-HT), via the 5-HT_2A receptor subtype, plays a key role in controlling the excitability of IHMNs. We hypothesized that in vivo 5-HT modulates IHMNs activity via the 5-HT_2A receptor subtype. To test this hypothesis, we used multibarrel micropipettes for extracellular single neuron recording and pressure picoejection of 5-HT or ketanserin, a selective 5-HT_2A receptor subtype antagonist, onto single IHMNs in decerebrate, vagotomized, paralyzed, and mechanically ventilated dogs. Drug-induced changes in neuronal discharge frequency (F_n) and neuronal discharge pattern were analyzed using cycle-triggered histograms. 5-HT increased the control peak F_n to 256 % and the time-averaged F_n to 340 %. 5-HT increased the gain of the discharge pattern by 61 % and the offset by 34 Hz. Ketanserin reduced the control peak F_n by 68 %, the time-averaged F_n by 80 %, and the gain by 63 %. These results confirm our hypothesis that in vivo 5-HT is a potent modulator of IHMN activity via the 5-HT_2A receptor subtype. Application of exogenous 5-HT shows that this mechanism is not saturated during hypercapnic hyperoxia. The two different mechanisms, gain modulation and offset change, indicate that 5-HT affects the excitability as well as the excitation of IHMNs in vivo.