Hypoglossal motoneurones play a major role in maintaining the patency of the upper airways and in determining airways resistance. These neurones receive inputs from many different regions of the neuroaxis including the caudal raphe nuclei. Whilst we have previously shown that glutamate is utilised in projections from one of these caudal raphe nuclei, the raphe pallidus, to hypoglossal motoneurones, these raphe pallidus-hypoglossal projections also contain multiple co-localised neuropeptides, including a population that are immunopositive for enkephalin. The role of enkephalin in the control of hypoglossal motoneurones is unknown. Therefore the aim of these studies was to determine whether enkephalins modulate caudal raphe glutamatergic inputs to hypoglossal motoneurones. Whole cell recordings were made from rat hypoglossal motoneurones in vitro, with glutamate-mediated excitatory postsynaptic currents (EPSCs) evoked in these neurones following electrical stimulation within the raphe pallidus. Superfusion of enkephalin significantly decreased the amplitude of these raphe pallidus evoked EPSCs (56.1+/-29% of control, P<0.001), an action that was mirrored by the tau-opioid receptor agonist, [D-Ala, N-Me-Phe, Gly-ol]-enkephalin acetate (DAMGO;53.8+/-26%, P<0.01), but not by the delta-opioid receptor agonist, [D-Pen]-enkephalin (DPDPE). Enkephalin also increased the amplitude ratio (1.57+/-0.36 vs. 1.14+/-0.27, P<0.01) of pairs of evoked EPSCs (paired pulse ratio), decreased the frequency (P<0.0001) but not the amplitude of miniature EPSCs, whilst having no effect on the inward current evoked by glutamate applied directly to the postsynaptic cell (97.8+/-2.2% of control, P=n.s.). Likewise, DAMGO also increased the paired pulse ratio (1.62+/-0.35 vs. 1.31+/-0.14, P<0.05) and decreased the frequency of miniature EPSCs (P<0.0001). Together, these data suggest that enkephalin acts at tau-opioid receptors located on the presynaptic terminals of raphe pallidus inputs to hypoglossal motoneurones to significantly decrease glutamate release from these projections.