EAAC1, a neuron-specific glutamate transporter, is likely to play an important role in the regulation of glutamate levels in the synaptic cleft. Ultrastructural studies have demonstrated that the glutamate receptor subunit proteins (e.g., GluR2) are frequently preferentially located at the postsynaptic density of asymmetric synapses. While the glutamate/glutamate receptor interaction is likely to be influenced by the activity and location of the transporter molecules, the spatial localization of the transporter molecules relative to the receptor molecules is not well delineated. Thus, we analyzed the cellular, ultrastructural, and synaptic distribution of EAAC1 in the context of the distribution of the AMPA receptor subunit GluR2 in the hippocampus. While GluR2 and EAAC1 are both present in hippocampal projection neurons, their intracellular distribution patterns differ. Both GluR2 and EAAC1 are present in the dendritic membranes and cytoplasm; however EAAC1 has a distinctive punctate distribution in the dendrite compared to the more diffuse labeling reflected by GluR2. Pre-embedding ultrastructural studies also revealed cytoplasmic and membrane-associated pools of EAAC1 within dendritic shafts and spines, as well as in a subset of axonal profiles and terminals. Postembedding double label immunogold localization demonstrated a similar intraneuronal distribution, but in addition showed that membrane-associated EAAC1 is not intermingled with GluR2 within the synaptic complex, but in contrast is primarily located perisynaptically, often immediately outside the synaptic specialization. In addition, there is a significant presynaptic pool of EAAC1, whereas GluR2 is essentially absent from the pre-synaptic profile. Thus, membrane-associated EAAC1 within the synaptic region is ideally situated to restrict the site of action of glutamate with respect to ionotropic receptors to the synaptic cleft, as well as regulate glutamate levels in the perisynaptic and presynaptic domains, the ultrastructural sites that have been associated with metabotropic receptor localization.