Left–right asymmetry is a fundamental feature of higher-order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluRε2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of ε2 subunits affects the properties of NMDARs and generates two populations of synapses, ‘ε2-dominant’ and ‘ε2-nondominant’ synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of β2-microglobulin (β2m)–deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well-known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity-dependent refinement of neuronal connections and normal synaptic plasticity. We found that β2m proteins were localised in hippocampal synapses in wild-type mice. NMDA EPSCs in β2m-deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25–6981, an ε2 subunit selective antagonist, with those in ‘ε2-dominant’ synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the β2m-deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of ‘ε2-dominant’ synapses. These observations indicate that the β2m-deficient hippocampus lacks ‘ε2-nondominant’ synapses and circuit 5 asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.