We have recently shown that the anticonvulsant drugs phenytoin, lamotrigine and sodium valproate all reduce the release of glutamate at synapses in the entorhinal cortex in vitro. In the present investigation we determined whether this property was shared by gabapentin and pregabalin, using whole-cell patch-clamp recordings of excitatory postsynaptic currents (EPSCs) in layer V neurons in slices of rat entorhinal cortex. Both drugs reduced the amplitude and increased the paired-pulse ratio of EPSCs evoked by electrical stimulation of afferent inputs, suggesting a presynaptic effect to reduce glutamate release. The frequency of spontaneous EPSCs (sEPSCs) was concurrently reduced by GBP, further supporting a presynaptic action. There was no significant change in amplitude although a slight reduction was seen, particularly with gabapentin, which may reflect a reduction in the number of larger amplitude sEPSCs. When activity-independent miniature EPSCs were recorded in the presence of tetrodotoxin, both drugs continued to reduce the frequency of events with no change in amplitude. The reduction in frequency induced by gabapentin or pregabalin was blocked by application of the l-amino acid transporter substrate l-isoleucine. The results show that gabapentin and pregabalin, like other anticonvulsants, reduce glutamate release at cortical synapses. It is possible that this reduction is a combination of two effects: a reduction of activity-dependent release possibly via interaction with P/Q-type voltage-gated Ca channels, and a second action, as yet unidentified, occurring downstream of Ca influx into the presynaptic terminals.