C2 spinal hemisection (C2Hx) results in inactivation of ipsilateral phrenic bursting due to interruption of bulbospinal respiratory pathways. Plasticity in the spinal phrenic circuitry results in partial recovery of phrenic output over weeks to months and the recovery correlates with time-dependent return of spinal serotonin (5-HT) immunoreactivity. Both the 5-HT type 2A and 2C receptor subtypes are present on phrenic motoneurons, and the 5-HT2A subtype is upregulated after C2Hx compared to spinal-intact tissues. We explored the functional role of these receptors by testing the hypothesis that pharmacologic blockade would attenuate phrenic motor output in rats with chronic (8-10 weeks) C2Hx. In initial experiments, bilateral phrenic activity was recorded in anesthetized, vagotomized and mechanically ventilated rats. The 5-HT2A/C receptor antagonist ketanserin (1 mg/kg, i.v.) had no impact on respiratory burst frequency, but triggered a persistent, bilateral reduction in phrenic burst amplitude in C2Hx but not spinal-intact rats. The relative reduction of phrenic bursting was considerably greater in the nerve ipsilateral to the lesion. In additional experiments, tidal volume and diaphragm EMG activity were measured during spontaneous breathing in vagally-intact anesthetized rats before and after i.v. ketanserin. The ipsilateral diaphragm EMG signal was significantly attenuated following ketanserin in C2Hx but not spinal-intact rats. The ketanserin-triggered reduction of ipsilateral diaphragm EMG was accompanied by a small increase in inspiratory tidal volume. We speculate that the increased tidal volume reflects robust compensatory recruitment of accessory inspiratory muscles. We conclude that activation of 5-HT2A/C receptors facilitates phrenic motoneuron bursting following chronic C2Hx, and that these receptors are likely located in the spinal cord.