1. With the use of whole-cell patch clamp recordings in slices of guinea-pig substantia gelatinosa (SG), we studied the serotonin (5-HT)- and noradrenaline (NA)-mediated inhibition of glutamate-mediated EPSCs evoked from primary afferent stimulation. 2. The frequency of spontaneous EPSPs was reduced by 5-HT and NA. 3. The inhibition of EPSCs caused by 5-HT was mediated by the 5-HT1D receptor subtype, since the 5-HT1D agonist, sumatriptan (1 microM), was effective. 4. NA and the alpha 2-agonist, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK 14304), decreased the EPSCs and this inhibition was blocked by the alpha 2-antagonists, idazoxan (1 microM) and yohimbine (1 microM). 5. The 5-HT-releasing agent, fenfluramine (10 microM), and the Na-releasing agent, amphetamine (1 microM), also depressed EPSCs. Pretreatment of slices with the 5-HT-depleting agent, p-chloro-amphetamine (10 microM), attenuated the inhibition of fenfluramine but failed to antagonize the effects of exogenously applied 5-HT. 6. These in vitro results suggest that presynaptic inhibition of glutamate release from primary afferents can provide another mechanism to explain the antinociceptive effects of 5-HT and NA obtained in vivo.