High-affinity uptake of glutamate from the synaptic cleft plays a crucial role in regulating neuronal activity in physiological and pathological conditions. We have used affinity-purified specific polyclonal antibodies raised against a synthetic peptide corresponding to the C-terminal region of rabbit and rat EAAC1, a glutamate (Glu) transporter believed to be exclusively neuronal, to investigate its cellular and subcellular localization and whether it is expressed exclusively in glutamatergic cells of infragranular layers, as suggested by previous studies. Light microscopic studies revealed that EAAC1 immunoreactivity (ir) is localized to neurons and punctate elements in the neuropil. EAAC1-positive neurons were more numerous in layers II-III and V-VI, i.e. throughout all projection layers. Most EAAC1-positive neurons were pyramidal, although nonpyramidal cells were also observed. Some EAAC1-positive non-pyramidal neurons stained positively with an antiserum to GAD, thus demonstrating that EAAC1 is not confined to glutamatergic neurons. Non-neuronal EAAC1-positive cells were also observed in the white matter, and some of them stained positively with an antiserum to GFAP. Ultrastructural studies showed that EAAC1-ir was in neuronal cell bodies, dendrites and dendritic spines, but not in axon terminals, i.e. exclusively postsynaptic. Analysis of the type of axon terminals synapsing on EAAC1-ir profiles showed that 97% of them formed asymmetric contacts, thus indicating that EAAC1 is located at the very sites of excitatory amino acid release. Unexpectedly, EAAC1-ir was also found in a few astrocytic processes located in both the gray and the white matter. The localization of EAAC1 may explain the pathological symptoms that follow EAAC knockout (seizures and mild toxicity), as seizures could be due to the loss of EAAC1-mediated fine regulation of neuronal excitability at axodendritic and axospinous synapses, whereas the mild toxicity may be related to the functional inactivation of astrocytic EAAC1.