Gamma-aminobutyric-acid (GABA) and ATP ionotropic receptors represent two structurally and functionally different classes of neurotransmitter-gated channels involved in fast synaptic transmission. We demonstrate here that, when the inhibitory rho1/GABA and the excitatory P2X2 receptor channels are co-expressed in Xenopus oocytes, activation of one channel reduces the currents mediated by the other one. This reciprocal inhibitory cross-talk is a receptor-mediated phenomenon independent of agonist cross-modulation, membrane potential, direction of ionic flux, or channel densities. Functional interaction is disrupted when the cytoplasmic C-terminal domain of P2X2 is deleted or in competition experiments with minigenes coding for the C-terminal domain of P2X2 or the main intracellular loop of rho1 subunits. We also show a physical interaction between P2X2 and rho1 receptors expressed in oocytes and the co-clustering of these receptors in transfected hippocampal neurons. Co-expression with P2X2 induces retargeting and recruitment of mainly intracellular rho1/GABA receptors to surface clusters. Therefore, molecular and functional cross-talk between inhibitory and excitatory ligand-gated channels may regulate synaptic strength both by activity-dependent current occlusion and synaptic receptors co-trafficking.