A semiquantitative electron microscopic immunocytochemical procedure was used to study the cellular and subcellular distribution of glutaraldehyde-fixed glutamate in rat hippocampal formation. Ultrathin plastic-embedded sections were incubated with a primary glutamate antiserum followed by a secondary antibody coupled to colloidal gold particles. A computer-assisted assessment of gold particle densities revealed that the axon terminals of all of the main excitatory pathways in the hippocampus were enriched with glutamate-like immunoreactivity relative to other tissue elements, including the parent cell bodies (granule and pyramidal cells). The different excitatory pathways showed slightly different labelling intensities: boutons in the termination zone of the lateral perforant path were covered by higher gold particle densities than boutons situated in the termination zones of the medial perforant path, the Schaffer collateral/commissural pathway and the hilar associational/commissural pathway. The mossy fibre terminals were significantly less enriched in immunoreactivity than terminals of the lateral perforant path and the Schaffer collateral/commissural pathway. Within the terminals, glutamate-like immunoreactivity was concentrated over synaptic vesicles and mitochondria. Terminals establishing symmetric junctions with cell bodies or dendritic stems displayed low particle densities, as did glial cell processes. These findings support the idea that glutamate is a major excitatory neurotransmitter in hippocampal excitatory synapses. Our observations are also in line with biochemical data pointing to the existence of a considerable neuronal and a smaller glial, metabolic pool of glutamate.