KCNQ subunits encode for the M current (I_KM), a neuron-specific voltage-dependent K⁺ current with a well established role in the control of neuronal excitability. In this study, by means of a combined biochemical, pharmacological, and electrophysiological approach, the role of presynaptic I_KM in the release of previously taken up tritiated norepineprine (NE), GABA, and d-aspartate (d-ASP) from hippocampal nerve terminals (synaptosomes) has been evaluated. Retigabine (RT) (0.01-30 μm), a specific activator of I_KM, inhibited [³H]NE, [³H]d-ASP, and [³H]GABA release evoked by 9 mm extracellular K⁺ ([K⁺]_e). RT-induced inhibition of [³H]NE release was prevented by synaptosomal entrapment of polyclonal antibodies directed against KCNQ2 subunits, an effect that was abolished by antibody preabsorption with the KCNQ2 immunizing peptide; antibodies against KCNQ3 subunits were ineffective. Flupirtine (FP), a structural analog of RT, also inhibited 9 mm [K⁺]_e-induced [³H]NE release, although its maximal inhibition was lower than that of RT. Electrophysiological studies in KCNQ2-transfected Chinese hamster ovary cells revealed that RT and FP (10 μm) caused a -19 and -9 mV hyperpolarizing shift, respectively, in the voltage dependence of activation of KCNQ2 K⁺ channels. In the same cells, the cognition enhancer 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 μm) blocked KCNQ2 channels and prevented their activation by RT (1-10 μm). Finally, both XE-991 (10-100 μm) and tetraethylammonium ions (100 μm) abolished the inhibitory effect of RT (1 μm) on [³H]NE release. These findings provide novel evidence for a major regulatory role of KCNQ2 K⁺ channel subunits in neurotransmitter release from rat hippocampal nerve endings.