Ionotropic excitatory amino acid (EAA) receptors are divided pharmacologically into three categories termed NMDA, AMPA/kainate, and high affinity kainate receptors. Each of these receptor subtypes is composed of a specific subset of subunits termed GluR1-4 (AMPA/kainate), GluR5-7, KA1-2 (high affinity kainate), and NMDAR1, 2 A-D (NMDA). Although colocalization of NMDA and non-NMDA receptors has been previously demonstrated electrophysiologically in rat, comprehensive analyses of subunit specific colocalization patterns have not been possible until the advent of appropriate antibodies. The present study investigates such immunocytochemical colocalization of several EAA receptor subunits within individual cells as well as dendritic spines in the monkey hippocampus. Double-label immunohistochemical experiments using antibodies which are specific for GluR2(4), GluR5-7, and NMDAR1 demonstrated that virtually all projection neurons in each subfield of the hippocampus contain subunits from the AMPA/kainate, kainate, and NMDA receptor families. In addition, confocal microscopy has demonstrated that individual spines may contain subunits representative of multiple EAA receptor families. Furthermore, detailed regional, cellular, and ultrastructural distribution patterns of the EAA receptor subunits GluR2 and GluR4 in monkey hippocampus are presented based on the use of a monoclonal antibody (mAb), 3A11, which was generated against the putative extracellular N-terminal domain of GluR2. Since this antibody recognizes only GluR2 in Western blots, and GluR2 as well as GluR4 in fixed transiently transfected cells, it has been designated anti-GluR2(4). Immunocytochemical labeling with mAb 3A11 revealed pyramidal cell somata and dendrites in each field of the hippocampus, as well as granule cells and polymorphic hilar cells in the dentate gyrus. Small cells with the morphologic characteristics of astroglia were also immunolabeled for GluR2(4) within the alveus and fimbria. Immunoreactivity at the ultrastructural level was localized to postsynaptic densities on dendritic spines and shafts and within the somatodendritic cytoplasm in all major hippocampal regions, as well as in a subset of dentate granule cell axons within the mossy fiber projection.