Impulses in primary afferent nerve fibers may produce short- or long-lasting modifications in spinal nociception. Here we have identified a robust long-term depression (LTD) of synaptic transmission in substantia gelatinosa neurons that can be induced by low-frequency stimulation of primary afferent Aδ-fibers. Synaptic transmission between dorsal root afferents and neurons in the substantia gelatinosa of the spinal cord dorsal horn was examined by intracellular recording in a transverse slice dorsal root preparation of rat spinal cord. Conditioning stimulation of dorsal roots with 900 pulses given at 1 Hz (10 V, 0.1 msec) produced LTD of EPSP amplitudes in substantia gelatinosa neurons to 41 ± 10% of control that lasted for at least 2 hr. When A- and C-fibers were recruited, conditioning stimulation was as effective as A-fiber stimulation alone. After LTD, synaptic strength could be increased to its original level by applying a second, high-frequency tetanic stimulus to the dorsal root, indicating that LTD is reversible and not attributable to damage of individual synapses. Bath application of the GABAA receptor antagonist bicuculline and glycine receptor antagonist strychnine did not affect LTD. When NMDA receptors were blocked by bath application ofd-2-amino-5-phosphonovaleric acid, LTD was abolished or strongly reduced. Loading substantia gelatinosa neurons with Ca2+ chelator BAPTA also blocked or reduced LTD. After incubation of slices with calyculin A, a selective and membrane permeable inhibitor of protein phosphatases 1 and 2A, LTD was not attenuated. We propose that this form of LTD may be relevant for long-lasting segmental antinociception after afferent stimulation.