Although exogenous serotonin at the hypoglossal motor nucleus (HMN) activates the genioglossus muscle, endogenous serotonin plays a minimal role in modulating genioglossus activity in awake and sleeping rats (Sood S, Morrison JL, Liu H, and Horner RL. Am J Respir Crit Care Med 172: 1338-1347, 2005). This result therefore implies that medullary raphe neurons also play a minimal role in the normal physiological control of the HMN, but this has not yet been established because raphe neurons release other excitatory neurotransmitters onto respiratory motoneurons in addition to serotonin. This study tests the hypothesis that inhibition of medullary raphe serotonergic neurons with 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) suppresses genioglossus and diaphragm activities in awake and sleeping rats. Ten rats were implanted with electrodes to record sleep-wake states and genioglossus and diaphragm activities. Microdialysis probes were also implanted into the nucleus raphe obscurus (NRO). Experiments in 10 anesthetized and vagotomized rats were also performed using the same methodology. In anesthetized rats, microdialysis perfusion of 0.1 mM 8-OH-DPAT into the NRO decreased genioglossus activity by 60.7+/-9.0% and diaphragm activity by 13.3+/-3.4%. Diaphragm responses to 7.5% CO2 were also significantly reduced by 8-OH-DPAT. However, despite the robust effects observed in anesthetized and vagotomized rats, there was no effect of 0.1 mM 8-OH-DPAT on genioglossus or diaphragm activities in conscious rats awake or asleep. The results support the concept that endogenously active serotonergic medullary raphe neurons play a minimal role in modulating respiratory motor activity across natural sleep-wake states in freely behaving rodents. This result has implications for pharmacological strategies aiming to manipulate raphe neurons and endogenous serotonin in obstructive sleep apnea.