Presynaptic GABAA receptor-mediated currents (34 °C) were recorded from mossy fiber boutons (MFBs) in rat hippocampal slices. In MFBs from young and adult animals, GABA puff- application activated currents which were blocked by GABAA receptor antagonists. The conductance density of 0.65 mS * cm2 was comparable to that of other presynaptic terminals. Single channel conductance was 36 pS (symmetrical chloride) yielding an estimated GABAA receptor density from 20 to 200 receptors per MFB. In accordance with the low apparent GABA affinity (EC50 60 mM) of the receptors and a tight control of ambient GABA concentration by GABA transporters, no tonic background activation of presynaptic GABAA receptors was observed. Instead, extracellular high frequency stimulation led to transient presynaptic currents, which were blocked by GABAA receptor antagonists, but were enhanced by block of the GABA transporter 1 indicating that these currents were generated by GABA spill- over and subsequent presynaptic GABAA receptor activation. Presynaptic spill-over currents were depressed by pharmacological cannabinoid 1 receptor activation. Since GABAA receptors in axons are considered to act depolarizing, high interneuron activity will exert substantial impact on presynaptic membrane potential, thus modulating action-potential- evoked transmitter release at the mossy fiber-CA3 synapse.