Glutamine is thought to play an integral role in the recycling pathway of glutamate. The glutamate-glutamine cycle proposes that perisynaptic astrocytes release glutamine which is then taken up by the presynaptic neuron as a glutamate precursor. Using fluorescent pHi measurement and whole-cell patch-clamp, we have investigated the transport mechanisms that may mediate this release of glutamine from astrocytes in situ. Brainstem slices containing the medial nucleus of the trapezoid body were obtained from P10-15 Wistar rats and perfused with aCSF containing glutamate, GABA and glycine receptor inhibitors. Astrocytes were whole-cell patch-clamped. Transporter substrates were dissolved in external solution and either applied via perfusate or by pressurised ejection from a puffer pipette. For imaging pHi, 0.5 mM 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) was included in the patch pipette and the fluorescence emission ratio measured following illumination at 465 and 405 nm. Values are means ± S.E.M., compared by ANOVA. System A (SA) transporter function was probed using the substrate inhibitors α-methyl-aminoisobutyric acid (MeAIB), alanine and serine, while system N (SN) function was isolated using its unique ability to transport Li+ in place of Na+. Histidine was used as a substrate inhibitor of both SA and SN. Puff application of glutamine (5 s) generated a membrane current (-20.0 ± 2.4 pA, n=21) which was blocked by bath applied alanine (10 mM; 1.6 ± 2.1 pA, p<0.05, n=8) and MeAIB (20 mM; -6.2 ± 2.1 pA, p<0.05, n=6). Substitution of external Na+ with Li+ abolished the glutamine current (0.4 ± 1.0 pA, p<0.05, n=6). MeAIB also blocked glutamine-induced currents after membrane conductance was reduced by substituting internal K+ with Cs+ (from -25.6 ± 7.3 pA to -1.9 ± 1.0 pA, p<0.05, n=6). Fluorescent pHi measurement revealed an alkalinisation during puff application of glutamine (ΔF/F0 = 0.27 ± 0.02, n=34). While bath application of histidine (20 mM) abolished the pH change (p<0.05, n=3), MeAIB only attenuated this change by 50 ± 5% (n=3; p<0.05) and serine (20 mM) blocked the pH change to a comparable extent (41 ± 8%; n=5; p<0.05). In addition, a substantial proportion of the pH change (63 ± 11%; n=7) was insensitive to the substitution of Na+ with Li+ (p<0.05). Together, these data identify two distinct glutamine transport systems within astrocytes. Properties of the glutamine-induced membrane currents indicate expression of SA while pH imaging reveals a second non-electrogenic glutamine transport system with properties identifying it as SN. Furthermore, the lack of effect of serine on SN transport suggests that the SN1 not the SN2 isoform predominates. SN is known to be readily reversible under physiological conditions and we propose that this mediates the astrocytic glutamine release mechanism within the glutamate-glutamine cycle.