1. This study was undertaken to gain more direct evidence of the pathways and neurochemical mediators of a spinally mediated baroreceptor inhibition of sympathetic preganglionic neurones (SPNs). 2. For this purpose, single-pulse electrical stimulation within identified vasodepressor regions of the nucleus tractus solitarii (NTS) was used together with extracellular recordings of single antidromically identified SPNs in the T2 segment of the spinal cord of anaesthetized rats. 3. The actions of agonists and antagonists of inhibitory amino acids on the NTS-induced inhibitions were determined, when they were iontophoretically applied in the vicinity of SPNs via a multibarrel micropipette assembly. 4. Extracellular recordings were made from sixty-nine SPNs. In forty-four SPNs, NTS stimulation elicited a period of inhibition of activity in both spontaneous and 'D,L-homocysteic acid-driven' SPNs with a latency to onset of 60 +/- 6 ms and a magnitude of 80 +/- 3%. 5. In six out of eight neurones, the NTS-induced inhibition was reduced by 74 +/- 16% during the application of the glycine antagonist strychnine (0-10 nA, 5-10 min) with doses that selectively blocked the inhibitory effect of iontophoretically applied glycine. 6. In nine out of nine neurones, the NTS-induced inhibition was reduced by 38 +/- 6% during the application of the GABAA antagonist bicuculline (5-15 nA, 4-14 min) with doses that selectively blocked the inhibitory effect of iontophoretically applied GABA. 7. In two SPNs, the actions of strychnine and bicuculline were shown to be additive in blocking the NTS inhibition. 8. The selective GABAB antagonists, CGP 35348 (20-50 nA, 6-25 min) and CGP 55845A (10 nA, 11 min) did not antagonize the NTS-induced inhibition. 9. It is suggested that GABA and glycine interneurones are activated by a baroreceptor bulbospinal pathway to inhibit sympathetic preganglionic neurones in the spinal cord.