The raphe pallidus (RPa) and Bötzinger complex (BötC) represent two important nuclei which project to spinal phrenic motor neurons. Stimulation of the RPa produces facilitative effects on respiratory activity, whereas stimulation of the BötC induces inhibitory effects on respiratory activity. In the present study, we examined the modulatory effects of serotonergic (5-hydroxytryptamine, 5-HT) RPa neurons on the inhibitory response of the phrenic nerve activity elicited from the BötC in rats. Experiments were performed on spontaneously breathing, urethane-anesthetized adult rats. Either high-frequency stimulation or glutamatergic chemical activation of the RPa region significantly attenuated the BötC-induced inhibition of the phrenic nerve. This attenuation showed a post-stimulation time and intensity dependency. Pharmacological experiments showed that intravenous injection of methysergide, a broad-spectrum antagonist of 5-HT receptors, markedly reduced the respiratory facilitation induced by electrical stimulation of the RPa. Furthermore, microinjections of methysergide into the cerebrospinal fluid around the phrenic motor nucleus (PMN) region at spinal cord segments C4 and C5 significantly decreased the RPa-related attenuation effects on BötC-evoked inhibition of phrenic nerve discharge. These results suggest that RPa serotonergic neurons could modulate the inhibition of phrenic nerve activity induced by BötC. Moreover, as the relevant 5-HT receptors for RPa's modulatory effects are located in the cervical spinal cord, 5-HT may, in part, function as a modulator to suppress the BötC neuronal activity via direct RPa-PMN and BötC-PMN convergent projection pathways to phrenic motoneurons.