Reported values of extracellular glutamate concentrations in the resting state depend on the method of measurement and vary ∼1000-fold. As glutamate levels in the micromolar range can cause receptor desensitization and excitotoxicity, and thus affect neuronal excitability, an accurate determination of ambient glutamate is important. Part of the variability of previous measurements may have resulted from the sampling of glutamate in different extracellular compartments, e.g., synaptic versus extrasynaptic volumes. A steep concentration gradient of glutamate between these two compartments could be maintained, for example, by high densities of glutamate transporters arrayed at the edges of synapses. We have used two photon laser scanning microscopy and electrophysiology to investigate whether extracellular glutamate is compartmentalized in acute hippocampal slices. Pharmacological blockade of NMDARs had no effect on Ca(2+) transients generated in dendritic shafts or spines of CA1 pyramidal neurons by depolarization, suggesting that ambient glutamate is too low to activate a significant number of NMDARs. Furthermore, blockade of transporters did not flood the synapse with glutamate, indicating that synaptic NMDARs are not protected from high concentrations of extrasynaptic glutamate. We suggest that, in the CA1 region of hippocampus, glutamate transporters do not create a privileged space within the synapse but rather keep ambient glutamate at very low levels throughout the neuropil.