Chronic dehydration induces structural changes in the hypothalamic supraoptic nucleus (SON), including increased glutamate synapses and retraction of astroglial processes. We performed whole-cell recordings in acute hypothalamic slices to determine whether these changes increase tonic activation of presynaptic metabotropic glutamate receptors (mGluRs) by increasing ambient glutamate in the SON. Activation of presynaptic group III mGluRs caused a decrease in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in SON neurones that was significantly attenuated in slices from dehydrated rats (-27.8 %) compared with untreated rats (-41.7 %), suggesting a higher basal occupancy of mGluRs by ambient glutamate during dehydration. Blocking group III mGluRs caused an increase in the frequency of mEPSCs that was significantly higher in slices from dehydrated rats (+42.8 %) than untreated rats (+31.4 %), suggesting greater tonic activation of presynaptic mGluRs by ambient glutamate during dehydration. Increasing ambient glutamate levels by inhibiting astrocyte glutamate uptake resulted in a decrease in mEPSC frequency due to increased activation of presynaptic mGluRs. This was attenuated in slices from dehydrated rats (-35.4 %) compared with slices from untreated rats (-48.8 %), suggesting diminished astrocytic glutamate uptake during dehydration. Immunochemical analyses revealed a robust expression of the GLT-1 transporter protein in the SON, which was diminished in SON punches from dehydrated rats compared with untreated controls. Thus, dehydration leads to increased tonic activation of presynaptic mGluRs on glutamate terminals, consistent with a decrease in glutamate buffering capacity. The resulting reduction in glutamate release probability may compensate for the increase in glutamate release sites that occurs during dehydration.