The lateral superior olivary nucleus (LSO) is an auditory relay centre within the brain stem that encodes interaural level differences for sound localization by integrating GABA/glycinergic input from the contralateral ear via the medial nucleus of the trapezoid body (MNTB), and glutamatergic input from the ipsilateral ear via the ventral cochlear nucleus (VCN). To study the development of the circuits that contribute to the establishment of sound localization, the heterosynaptic modulation mediated by glutamate released from VCN terminals and group II metabotropic glutamate receptor (mGluR) expressed on MNTB inhibitory terminals was investigated using whole-cell patch-clamp techniques. At postnatal day-4-8 (P4-8), repetitive stimulation of the VCN-LSO excitatory afferents caused significant inhibition of MNTB-LSO inhibitory postsynaptic currents (IPSCs) in amplitude with an increase of its coefficient of variation and changed the paired-pulse ratio. These effects were antagonized by LY341495, an mGluR2/3 antagonist. Thus, the suppression of MNTB-LSO synaptic responses induced by repetitive stimulation applied to the VCN-LSO glutamatergic afferent is presumably due to an activation of mGluR2/3 existing on MNTB-LSO presynaptic terminals. The suppression rate of MNTB-LSO IPSCs by DCG IV, an mGluR2/3 agonist, decreased with development and became negligible by the third week after birth. The immunohistochemical staining of mGluR2/3 in the LSO was also less apparent at P18 compared with that at P4. We suggest that mGluR-mediated heterosynaptic modulation of MNTB-LSO GABAergic/glycinergic transmission might contribute to the development of appropriate adult auditory circuits.