Abstract In these studies we determined the role of the medullary midline nuclei on a cholinergic outflow to the airways by examining the response of tracheal tone and lung resistance to pharmacological stimulation. Studies were performed on α-chloralose-anesthesized, paralyzed and mechanically ventilated cats, and ferrets. l-glutamate microinjection into the medullary midline neurons significantly decreased tracheal tension, and reduced lung resistance. These effects were abolished by prior topical application of methysergide, a broad spectrum serotonin receptor antagonist. Stimulation of the medullary raphe nuclei was also associated with a significant increase in the phrenic nerve output, and a decrease in arterial blood pressure. The results indicate that the medullary midline neurons are involved in regulation of cholinergic outflow to the airways, and raise the possibility that alterations in the serotonergic pathways may cause airway dysfunction.