The ephrin receptors EphA4 and EphB2 have been implicated in synaptogenesis and long-term potentiation in the cerebral cortex and hippocampus, where they are generally viewed as post-synaptic receptors. To determine the precise distribution of EphA4 and EphB2 in mature brain synapses, we used subcellular fractionation and electron microscopy to examine the adult mouse forebrain/midbrain. EphA4 and EphB2 were both enriched in microsomes and synaptosomes. In synaptosomes, they were present in the membrane and the synaptic vesicle fractions. While EphA4 was tightly associated with PSD-95-enriched post-synaptic density fractions, EphB2 was easily extracted with detergents. In contrast, both receptors were found in the pre-synaptic active zone fraction. By electron microscopy, EphA4 was mainly detected in axon terminals, whereas EphB2 was more frequently detected in large dendritic shafts, in the hippocampus and cerebral cortex. However, in the ventrobasal thalamus, EphB2 was detected most frequently in axon terminals and thin dendritic shafts. The localization of EphA4 and EphB2 in multiple compartments of neurons and synaptic junctions suggests that they interact with several distinct scaffolding proteins and play diverse roles at synapses.