Non-N-methyl-D-aspartate glutamate receptors are responsible for fast excitatory neurotransmission in the mammalian CNS. These receptors are rapidly activated and desensitized in the presence of glutamate, and are often further subdivided into alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid and kainate receptors based on their selective agonists. Non-NMDA glutamate receptors are composed of multiple subunits which recently have been cloned, and studies on the recombinant glutamate receptors have helped clarify the distinctions between AMPA and kainate-preferring glutamate receptors. Although the subunits which make up both AMPA and kainate receptors have a widespread distribution, most currents recorded in vivo are characteristic of recombinant AMPA receptors. To help clarify the functional role of high-affinity kainate receptors, we have characterized the expression of a high-affinity kainate receptor subunit, KA2, in cultured hippocampal neurons. Using immunocytochemistry, we found that KA2 was expressed in hippocampal neurons at all times during the development of the cells in culture, and the subunit was enriched in dendritic spines after about 14 days. The subcellular distribution of KA2 paralleled that of the AMPA receptor subunit GluR1, with the AMPA and kainate subunits being colocalized at all times in culture. The enriched KA2 immunoreactivity co-localized with the synaptic vesicle protein synaptophysin at the resolution of light microscopy, indicating synaptic localization of KA2. Although the kainate subunit KA2 co-localized with the AMPA subunit GluR1, co-immunoprecipitation experiments demonstrated a direct interaction between the AMPA receptor subunits GluR1 and GluR2/3, but not between GluR1 and the kainate subunits GluR6/7 or KA2. We therefore, conclude that both AMPA and kainate receptor subunits are enriched in the same dendritic spines, yet do not combine to form receptor complexes.