Metabotropic GABA(B) receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here we tested the hypothesis that glutamatergic neurotransmission may feed back to regulate GABA(B) receptors. We found that application of glutamate to cultured cortical neurons led to rapid downregulation of GABA(B) receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by co-activation of group I metabotopic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca2+ channels and removal of extracellular Ca2+ prevented glutamate-induced downregulation of GABA(B) receptors, indicating that Ca2+-influx plays a critical role. We further established that glutamate-induced downregulation depends on the internalization of GABA(B) receptors. Glutamate did not affect the rate of GABA(B) receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA(B) receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors downregulates GABA(B) receptors by sorting endocytosed GABA(B) receptors preferentially to lysosomes for degradation on expense of recyling. This mechanism may relieve glutamatergic synapses from GABA(B) receptor-mediated inhibition resulting in increased synaptic excitability.