1. The nucleus tractus solitarius (NTS) is the first central site where the reflex control of autonomic, including baroreceptor, reflex function is coordinated. Autonomic signals are transmitted from the first-order visceral afferent fibres to second-order NTS neurons by L-glutamate. It is well established that activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, which mediate the fast component of L-glutamate signalling, is required for generating changes in membrane potentials of the second-order NTS neurons. The contribution of the slower-developing, longer-lasting N-methyl-D-aspartate (NMDA) receptor-mediated component of glutamate signalling to synaptic transmission at these synapses is less well understood. 2. The aim of this work is to highlight evidence that functional NMDA receptors exist on second-order NTS neurons in autonomic, including baroreceptor, afferent pathways by determining whether NMDA receptors can be activated by: (i) exogenous application of NMDA; and (ii) endogenous release of L-glutamate from autonomic afferent fibres. Studies were performed on second-order neurons in transverse and horizontal brainstem slices containing the intermediate NTS and the tractus solitarius. Second-order NTS neurons were identified by electrophysiological criteria or by attached fluorescent-labelled aortic depressor nerve (ADN) boutons. 3. N-Methyl-D-aspartate (50 nmol(-2) micromol) dose-dependently evoked excitatory post-synaptic currents in second-order NTS neurons (P = 0.004; n = 4). The NMDA receptor-mediated currents were also synaptically evoked by low-frequency stimulation of the autonomic afferent fibres in the tractus solitarius. The NMDA receptor-mediated currents were blocked by the NMDA receptor antagonist AP5 (n = 7; P = 0.027). 4. The findings suggest that functional NMDA receptors exist on second-order NTS neurons. While the NMDA receptor- mediated currents may not be required for signal transmission when the second-order neurons are at resting membrane potential, their activation may help to modulate autonomic signal transmission in the NTS under conditions in which the membrane is depolarized by high frequency or convergent inputs.