Glutamate is the main excitatory neurotransmitter in the mammalian CNS. It has been proposed that presynaptic terminals sequester glutamine from the extracellular fluid to replenish synaptically released glutamate. Using direct electrophysiological recordings from the calyx of Held presynaptic terminal, we investigated the nature of the presynaptic glutamine transport mechanism and assessed its role in maintaining glutamatergic synaptic transmission. Brainstem sections containing the medial nucleus of the trapezoid body (MNTB) were obtained from P10-15 Wistar rats. Presynaptic terminals were whole-cell voltage-clamped and glutamine transporter currents were elicited by 5 s puff-application of 10 mM glutamine from an adjacent pipette. Values are means ± S.E.M., compared by ANOVA. In quiescent presynaptic terminals the glutamine induced current (Igln) was almost absent (-0.7 ± 0.4 pA; n=17). However, activation of Ca2+ currents in the presynaptic terminal by depolarisation (-80 to 0 mV for 2 ms, repeated at 100 Hz for 2 s) consistently resulted in the rapid establishment of a robust Igln (-12.9 ± 1.6 pA; n=24). The induction of Igln was blocked by the removal of external Ca2+ (by 93 ± 3 %; n=3; p<0.001) and was inhibited by inclusion of botulinum neurotoxin C in the patch-pipette (by 57 ± 2 %; n=3; p<0.05), indicating the involvement of vesicle fusion. Cessation of presynaptic stimulation resulted in down-regulation of Igln with a half-time of <30 s. Furthermore, Igln was eliminated by the removal of external Na+ (by 99 ± 1%; n=3; p<0.001) and by the specific system A neutral amino acid transport inhibitor α-methyl-aminoisobutyric acid (MeAIB; reduced by 94 ± 3 %; n=3; p<0.001). At depolarised potentials Igln did not reverse, consistent with activation of a transporter. Additionally, puff application of 200 μM glutamate did not induce a significant membrane current (0.0 ± 0.5 pA; n=3; p<0.05), indicating a lack of direct presynaptic glutamate uptake. Miniature EPSCs recorded from the postsynaptic MNTB principal neurons (-38.8 ± 3.5 pA) were inhibited by MeAIB (-28.6 ± 3.4 pA; n=5; p<0.01) following stimulation at 200 Hz to promote turnover of the presynaptic glutamate pool. Single EPSCs in the absence of high frequency stimulation were unaffected by MeAIB (-4.5 ± 0.5 nA in control and -4.3 ± 0.4 nA in MeAIB; n=3 p=0.4), indicating that MeAIB does not have a direct effect on postsynaptic AMPA receptors and is acting to reduce vesicular glutamate content. These data indicate that system A mediated glutamine transport occurs in glutamatergic nerve terminals and that its functional expression in the presynaptic plasma membrane is dynamically controlled by synaptic activity. They also indicate that system A is important in maintaining the supply of glutamate for excitatory neurotransmission during periods of high frequency stimulation.