An understanding of the neural control of lower oesophageal sphincter (LOS) relaxation is clinically relevant because transient LOS relaxations (TLOSRs) are a mechanism of acid reflux into the oesophagus. Preganglionic motor neurones innervating the LOS are localized in the dorsal motor nucleus of the vagus (DMV). Based on a single study in cats, it is now widely accepted that these neurones are functionally organized into two separate populations, such that stimulation of the caudal and rostral DMV evokes LOS relaxation and contraction, respectively. Our goal was to map the functional LOS responses to chemical stimulation in the DMV and nucleus tractus solitarius (NTS) of ferrets, an animal model commonly used for conscious studies on TLOSRs, and to test whether DMV-evoked LOS relaxation is mediated through hexamethonium-sensitive vagal-inhibitory pathways to the LOS. We used miniaturized manometry with Dentsleeve to monitor LOS and oesophageal pressures in decerebrate unanaesthetized ferrets. LOS relaxation was evoked readily in response to gastric insufflation, which shows that the vago-vagal reflex was intact in this preparation. Microinjections of l-glutamate (12.5 nmol L-1 in 25 nL) were made into the DMV from approximately - 1.5 to + 2.0 mm relative to the obex. Microinjections into the caudal (- 1.5 to + 0.0 mm behind obex) and intermediate (+ 0.1 to + 1.0 mm rostral to obex) DMV both significantly decreased LOS pressure, and complete LOS relaxation was noted in 28/32 and 11/18 cases, respectively. LOS relaxation responses to DMV microinjection were highly reproducible and abolished by bilateral vagotomy or hexamethonium (15 mg kg-1 intravenously). A nitric oxide synthase inhibitor (l-NAME 100 mg kg-1 intramuscularly) significantly increased the time taken to reach the maximal response. Increases in LOS pressure (24 +/- 4 mmHg; n = 3) were obtained only when stimulation sites were located equal to greater than 1.5 mm rostral to the obex. LOS relaxation (- 78 +/- 10%; n = 6) was evoked by stimulation of the NTS but not immediately outside of the NTS (11 +/- 27%; n = 5). We conclude that there is a very extensive population of 'inhibitory' motor neurones in the DMV that may account for the predominant vagal-inhibitory tone in ferrets. As NTS stimulation evokes LOS relaxation and the predominant response to DMV stimulation is also LOS relaxation, this vago-vagal reflex may involve an excitatory interneurone between the NTS and DMV vagal inhibitory output.