Glutamate has a high concentration within the placental syncytiotrophoblast (5 mmol/l-1) but its role is uncertain. Glutamate uptake is mediated by System XAG but it remains unclear how it is released from the placental syncytiotrophoblast. The aim of this study was to investigate mechanisms by which glutamate might be released from the microvillous membrane (MVM) of the placental syncytiotrophoblast into the maternal circulation. Ethical approval for this study was granted by the Southampton and Southwest Hampshire Regional Ethics Committee. Placentas were collected following uncomplicated term pregnancies. Isolated perfused human placental cotyledons (n = 10) were perfused with modified Earle’s bicarbonate buffer. 3H-proline, 14C-glutamate and 1.8 mM creatinine were perfused into the maternal arterial circulation. A bolus (16 µmol) of glutamate and a bolus of the System Xc- substrate, N-acetylcysteine (0.1 mol), were injected into the circulation to stimulate glutamate transport by exchange. In addition, following the cessation of isotope perfusion, a bolus of 50 mmol urea was added to the maternal circulation to create an osmotic shock. Radioactivity in maternal and fetal venous samples were determined via liquid scintillation counting and creatinine levels were determined using an enzymatic assay. PCR was used to identify whether Xc-, OAT1, OAT2, OAT4, OAT5 and OAT8 were expressed in the placenta. Western blotting was used to determine whether Xc- was localised to the MVM. The maternal N-acetylcysteine bolus stimulated release of glutamate, but not proline, into the maternal circulation (n = 5). PCR confirmed the expression of Xc- in human cytotrophoblast tissue samples and western blotting localised this to the MVM. Following the maternal arterial urea bolus there was release of glutamate but not proline into the maternal circulation (n = 5).This study provides evidence for two routes of glutamate efflux from the placental syncytiotrophoblast into the maternal circulation. The first is via the amino acid exchanger System Xc- which was localised to the MVM. Activity of System Xc- is a rate limiting step in the provision of cystine to the cell for glutathione synthesis. As a result, high intracellular glutamate levels may create a gradient which mediates the uptake of cystine for glutathione production, protecting the fetus from oxidative stress. The release of glutamate in response to an osmotic shock is consistent with the volume regulated anion channel. This channel may be involved in regulating the volume of the syncytiotrophoblast. These data suggest that high intracellular glutamate concentrations generate a gradient which can be used to drive membrane transport and maintain cellular homeostasis.