Substance P modulates reflex regulation of respiratory function by its actions both peripherally and in the CNS, particularly in the nucleus tractus solitarius (NTS), the first central site for synaptic contact of the lung and airway afferent fibres. There is considerable evidence that substance P actions in the NTS augment respiratory reflex output, but the precise effects on synaptic transmission have not been determined. Therefore, we determined the effects of substance P on synaptic transmission at the first central synapses by using whole-cell voltage-clamping in an NTS slice. Studies were performed on second-order neurons anatomically-identified as receiving monosynaptic input from sensory nerves in the lungs and airways by fluorescent attached boutons labeled by tracheal instillation of 1,1'-dioctadecyl3,3,3',3' tetra- methylindocarbo-cyanine perchlorate (DiI). Substance P (1.0, 0.3 and 0.1 µ) in a concentration-dependent manner significantly decreased the amplitude of excitatory postsynaptic currents (eEPSCs) evoked by stimulation of the tractus solitarius. The decrease was accompanied by an increase in the paired pulse ratio of two consecutive evoked eEPSCs, and a decrease in the frequency, but not amplitude of spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs), findings consistent with a presynaptic site of action. The effects were consistently and significantly attenuated by a neurokinin-1 (NK1) receptor antagonist, (SR 140333, 3 µ). The data suggest a new site of action for substance P in the NTS (NK1 receptors on the central terminals of sensory fibres) and a new mechanism (depression of synaptic transmission) for regulating respiratory reflex function.