Little is known about which ion channels determine the resting electrical properties of presynaptic membranes. In recordings made from the rat calyx of Held, a giant mammalian terminal, we found resting potential to be controlled by KCNQ (Kv7) K(+) channels, most probably KCNQ5 (Kv7.5) homomers. Unlike most KCNQ channels, which are activated only by depolarizing stimuli, the presynaptic channels began to activate just below the resting potential. As a result, blockers and activators of KCNQ5 depolarized or hyperpolarized nerve terminals, respectively, markedly altering resting conductance. Moreover, the background conductance set by KCNQ5 channels, together with Na(+) and hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels, determined the size and time course of the response to subthreshold stimuli. Signaling pathways known to directly affect exocytic machinery also regulated KCNQ5 channels, and increase or decrease of KCNQ5 channel activity controlled release probability through alterations in resting potential. Thus, ion channel determinants of presynaptic resting potential also control synaptic strength.