Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I–IIo showed internalization. This internalization was inhibited by GABA (100 μm) and the GABAB agonist R-baclofen (10 μm), but not by the GABAA agonist muscimol (20 μm) or glycine (100 μm). The GABAB antagonist 2-hydroxysaclofen (100 μm) reversed the inhibitory effect of GABA, but not the GABAA antagonist bicuculline (100 μm). These findings demonstrate that GABAB receptors, but not GABAA or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 μm), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABAB receptors. In the presence of the GABAB antagonist 2-hydroxysaclofen (100 μm), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 μm) or the channel blocker MK-801 (10 μm). Therefore, inhibition of substance P release by GABAB receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.